BACKGROUND: Biomedical HIV prevention trials enroll sexually active women at risk of HIV and often discontinue study product during pregnancy. We assessed risk factors for pregnancy and HIV acquisition, and the effect of pregnancy on time off study drug in HIV Prevention Trials Network 039. METHODS: A total of 1358 HIV negative, herpes simplex virus type 2-seropositive women from South Africa, Zambia, and Zimbabwe were enrolled and followed for up to 18 months. RESULTS: A total of 228 pregnancies occurred; time off study drug due to pregnancy accounted for 4% of woman-years of follow-up among women. Being pregnant was not associated with increased HIV risk (hazard ratio 0.64, 95% confidence interval 0.23-1.80, P = 0.40). However, younger age was associated with increased risk for both pregnancy and HIV. There was no association between condom use as a sole contraceptive and reduced pregnancy incidence; hormonal contraception was not associated with increased HIV risk. Bacterial vaginosis at study entry was associated with increased HIV risk (hazard ratio 2.03, P = 0.02). CONCLUSIONS:: Pregnancy resulted in only a small amount of woman-time off study drug. Young women are at high risk for HIV and are an appropriate population for HIV prevention trials but also have higher risk of pregnancy. Condom use was not associated with reduced incidence of pregnancy.