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Adolescents may be appropriate for inclusion in biomedical HIV prevention trials. Adolescents' overall willingness to participate (WTP) in biomedical HIV prevention trials was examined, including after the prematurely discontinued phase IIb HVTN 503/Phambili HIV vaccine trial, in Soweto, South Africa.An interview-administered cross-sectional survey was conducted among 506 adolescents (16-18 years) between October 2008 and March 2009. The assessment included WTP in HIV prevention trials, sexual and substance use behavior, and related psychosocial constructs. Multivariate logistic regression analyses examined predictors of WTP in biomedical prevention trials.The sample primarily consisted of female participants (n = 298, 59%), and 50% of all participants were sexually active. WTP in general was high (93%), with 75% WTP in a vaccine trial after being informed about the HVTN 503/Phambili trial. Less exposure to stressors [odds ratio (OR): 2.8, confidence interval (CI): 1.3 to 6.3] was associated with adolescents' WTP in HIV biomedical prevention trials overall. Those with less exposure to stressors (OR: 1.7, CI: 1.1 to 2.8) and not sexually active (OR: 2.1, CI: 1.4 to 3.3) were predictive of WTP after the HVTN 503/Phambili trial. A higher number of sexual partners were associated with unwillingness to participate more generally (P = 0.039) and specifically after the HIV vaccine trial (P = 0.0004).The high level of adolescents' WTP in biomedical prevention trials is encouraging, especially after the prematurely discontinued HVTN 503/Phambili HIV vaccine trial. High-risk youth were less likely to be WTP, although those not yet sexually active were more WTP. Future biomedical HIV prevention trials should address challenges to enrollment of high-risk adolescents who may show less WTP. [ABSTRACT FROM AUTHOR]