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Undiagnosed HIV infection remains a significant public health problem. To address this, the Centers for Disease Control and Prevention revised testing recommendations, calling for routine opt-out HIV screening among adults in health care settings. However, these recommendations have not been widely implemented in primary care settings. We examined acceptability of opt-out H1V testing in an urban community health center and factors associated with accepting testing. From July 2007 to March 2008, physicians or a designated HIV tester approached patients presenting for primary care visits during 52 clinical sessions at an urban community health center. Patients were told they 'would be tested for HIV unless they declined testing.' Enzyme-linked immunosorbent assays, which required venipuncture, were used to test for HIV infection. We extracted demographic, clinical, and visit characteristics from medical records and examined associations between these characteristics and accepting HIV testing using logistic regression. Of 300 patients, 35% agreed to HIV testing, with no new HIV infections detected. Common reasons for declining testing were perceived low risk (54.4%) and self-reported HIV testing previously (45.1%). Younger age (adjusted odds ratio [AOR] = 0.97, 95% confidence interval [CI] = 0.96-0.99), Hispanic ethnicity (AOR = 1.78, 95% CI = 1.01-3.14), and having another blood test during the visit (AOR = 6.36, 95% CI = 3.58-11.28) were independently associated with accepting H1V testing. This study questions whether expanding HIV testing by conducting routine opt-out HIV testing in primary care settings is an acceptable strategy. It is important to understand how various testing strategies may affect HIV testing rates. In addition, further exploration of patients' reasons for declining HIV testing in these settings is warranted.