OBJECTIVES: To estimate the incidence of herpes simplex type 2 virus (HSV-2) infection, to identify risk factors for its acquisition, and to assess the protective effect of condoms. STUDY DESIGN: Prospective study of 293 HSV-2 seronegative women, aged 18 to 35 years, attending a sexually transmitted disease clinic in Alabama from 1992 to 1995. RESULTS: Incidence of HSV-2 infection was 20.5 per 100 woman-years [95% confidence interval (CI), 13.1-30.5]. Young women (18-20 years) had a significantly higher risk of incident HSV-2 infection [adjusted hazard ratio (HR), 2.8; 95% CI, 1.3-6.4] than older women. Women diagnosed with prevalent or incident bacterial vaginosis had a higher incidence of HSV-2 infection than those who were not so diagnosed (adjusted HR, 2.4; 95% CI, 1.1-5.6). No significant protective effect was observed for consistent (100%) condom use without breakage and slippage against HSV-2 acquisition (adjusted HR, 0.8; 95% CI, 0.2-2.3). CONCLUSION: Acquisition of HSV-2 infection among study participants was higher than previous estimates for adult female sexually transmitted disease clinic attendees, and no protective effect for condoms was demonstrated. The high incidence of HSV-2 infection with its potential for adverse health consequences emphasizes the need for better prevention strategies. © 2008, American Sexually Transmitted Diseases Association.