A longitudinal cohort study of sexually active women 18–30 years of age was conducted to identify variables associated with the acquisition of herpes simplex virus type 2 (HSV-2) infections. Six hundred seventy HSV-2–seronegative women were followed up at 4-month intervals for 1 year; acquisition of HSV-2 antibodies was detected in 32 of these women. Black race, ⩽12 years of education, having a new sex partner, and bacterial vaginosis (BV) were associated with HSV-2 seroconversion on univariate analysis. Antecedent HSV-1 infection was not protective against HSV-2 acquisition. After controlling for other identified risk factors in multivariable models, the diagnosis of BV remained associated with an increased risk of acquiring HSV-2 infection (hazard ratio, 2.1; 95% confidence interval, 1.0–4.5; P = .05). In this study, the population attributable risk of BV for HSV-2 seroconversion was 21%. Additional studies are needed to determine whether screening and treatment of BV could reduce susceptibility to the acquisition of HSV-2 in women.