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This study investigated the impact of prevalent and incident HSV-2 infection on the incidence of HIV-1 infection in a cohort of female commercial sex workers in KwaZulu-Natal, South Africa. Prior to a vaginal microbicide trial, 416 women were screened for antibodies to HIV-1 and herpes simplex virus-2 (HSV-2) infections and a questionnaire was used to establish behavioral, social, and demographic characteristics. A total of 187 HIV-1-seronegative women were followed up at monthly intervals when blood was drawn and used to detect HIV-1 and HSV-2 antibodies. The median duration of follow-up was 2.2 years. At screening 50% of the women were HIV-1 seropositive and 84% were HSV-2 seropositive. The hazards of HIV-1 among women who were HSV-2 seropositive or seronegative throughout, or among those who seroconverted during the study, were not significantly different. When HSV-2 seroconversion was analyzed as a time-dependent covariate, the hazard ratio for HIV-1 seroconversion was 6.0 (95% CI: 2.6-14.0) times greater among women with incident than among women with prevalent HSV-2 infections. Drawing on other recent studies these data suggest that incident HSV-2 infection increases the risk of HIV-1 infection; the effect wanes with time since infection; and the effect is significantly greater for men than it is for women.