Background The Botswana government recently implemented a policy of routine or “opt-out” HIV testing in response to the high prevalence of HIV infection, estimated at 37% of adults. Methods and Findings We conducted a cross-sectional, population-based study of 1,268 adults from five districts in Botswana to assess knowledge of and attitudes toward routine testing, correlates of HIV testing, and barriers and facilitators to testing, 11 months after the introduction of this policy. Most participants (81%) reported being extremely or very much in favor of routine testing. The majority believed that this policy would decrease barriers to testing (89%), HIV-related stigma (60%), and violence toward women (55%), and would increase access to antiretroviral treatment (93%). At the same time, 43% of participants believed that routine testing would lead people to avoid going to the doctor for fear of testing, and 14% believed that this policy could increase gender-based violence related to testing. The prevalence of self-reported HIV testing was 48%. Adjusted correlates of testing included female gender (AOR = 1.5, 95% CI = 1.1–1.9), higher education (AOR = 2.0, 95% CI = 1.5–2.7), more frequent healthcare visits (AOR = 1.9, 95% CI = 1.3–2.7), perceived access to HIV testing (AOR = 1.6, 95% CI = 1.1–2.5), and inconsistent condom use (AOR = 1.6, 95% CI = 1.2–2.1). Individuals with stigmatizing attitudes toward people living with HIV and AIDS were less likely to have been tested for HIV/AIDS (AOR = 0.7, 95% CI = 0.5–0.9) or to have heard of routine testing (AOR = 0.59, 95% CI = 0.45–0.76). While experiences with voluntary and routine testing overall were positive, 68% felt that they could not refuse the HIV test. Key barriers to testing included fear of learning one's status (49%), lack of perceived HIV risk (43%), and fear of having to change sexual practices with a positive HIV test (33%). Conclusions Routine testing appears to be widely supported and may reduce barriers to testing in Botswana. As routine testing is adopted elsewhere, measures should be implemented to assure true informed consent and human rights safeguards, including protection from HIV-related discrimination and protection of women against partner violence related to testing., Editors' Summary Background. In 2005, there were 5 million new infections with the human immunodeficiency virus (HIV), and the disease it causes—acquired immunodeficiency syndrome (AIDS)—killed three million people. Despite the increased availability of drugs that can fight HIV (antiretrovirals), the AIDS epidemic continues to grow, particularly in sub-Saharan Africa. To halt it, more needs to be done to prevent the spread of HIV. Education about safe sex can help—HIV is most commonly spread through unprotected sex with an infected partner—but increasing HIV testing is of paramount importance. Unfortunately, the uptake of voluntary counseling and testing in sub-Saharan Africa is worryingly low. Fear of being stigmatized—socially disgraced—and discriminated against, fears about the positive result itself, and worries about access to antiretroviral drugs are all putting people off being tested. Why Was This Study Done? In Botswana, one in three adults is infected with HIV. Since 2002, antiretroviral drugs have been freely available but enrollment in the Botswana National Treatment Program during its first two years was slow, in part due to inadequate uptake of voluntary HIV testing. Consequently, in early 2004, the government introduced a policy of routine HIV testing in which all patients are tested for HIV when they visit their doctor unless they opt out. A major aim of this approach to HIV testing, which was formally recommended in June 2004 by UNAIDS and the World Health Organization, is to increase uptake of HIV testing and treatment, and to reduce HIV-related stigma by treating the HIV test like any other routine medical procedure. However, there are fears that the policy could back-fire—people might not visit their doctors, for example, because they are afraid of being tested and think that they will not be able to refuse the test. In this study, the researchers investigated knowledge of and attitudes to routine testing in Botswana to understand better the consequences of a routine testing policy. What Did the Researchers Do and Find? The researchers interviewed adults throughout Botswana about their knowledge of and attitudes to routine HIV testing 11 months after introduction of the policy. Only half of the participants had heard of routine testing before being interviewed but nearly all were in favor of routine testing. More than half thought it would reduce HIV-related stigma and the violence toward women that is associated with an HIV-positive status. However, almost half believed that routine testing might prevent people from going to the doctor because of fear of testing and a few thought the policy would increase violence against women. Nearly half of the interviewees had had an HIV test and the researchers found, for example, that women were more likely to have been tested than men and that people with stigmatizing attitudes toward people living with HIV and AIDS were less likely to be tested. Fear of learning one's HIV status, lack of perceived risk, and fear of having to change sexual practices if positive all stopped people taking the test. Finally, although experiences with testing were generally positive, approximately two-thirds of interviewees who had been tested felt that it would have been difficult to refuse the test. What Do These Findings Mean? These results show that there is widespread support for routine HIV testing in Botswana, a finding supported by recent increases in treatment uptake. Routine testing, write the researchers, holds significant promise for the prevention and treatment of HIV/AIDS in Botswana and elsewhere. In particular, increasing the number of people tested for HIV may reduce HIV-related stigma, which should further increase testing and hopefully slow the spread of HIV. But the results of this study also highlight some areas of concern. Whenever HIV testing policies are implemented, human rights must be protected by ensuring that patients have all the information necessary to make an informed and free decision about being tested, by providing protection for women against violence related to HIV status, and by ensuring total confidentiality. Careful monitoring of Botswana's program and similar programs will be needed to ensure that these human rights are fully met, conclude the researchers. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030261 • US National Institute of Allergy and Infectious Diseases factsheet on HIV infection and AIDS • US Department of Health and Human Services information on AIDS • US Centers for Disease Control and Prevention information on HIV/AIDS • UNAIDS and World Health Organization 2004 policy statement on HIV testing • AVERT, a UK-based charity, provides information about HIV and AIDS in Botswana, A cross-sectional, population-based study of 1,268 adults from five districts in Botswana showed that routine HIV testing appears to be widely supported and may reduce barriers to HIV testing.