To assess the timing of symptomatic genital ulcer disease (GUD) relative to human immunodeficiency virus (HIV) seroconversion, we studied 248 case subjects who underwent HIV seroconversion and 496 HIV-negative control subjects, at 3 interview visits conducted at 10-month intervals: visit 1, before HIV acquisition; visit 2, after seroconversion; and visit 3, 10 months after detection of seroconversion. Odds ratios (ORs) and 95% confidence intervals (CIs), for HIV acquisition, were estimated by logistic regression. HIV load was measured by RNA-polymerase chain reaction, and herpes simplex virus type 2 (HSV-2) serologic testing used HerpeSelect EIA with Western blot confirmation. The OR of HSV-2 seropositivity associated with HIV acquisition was 1.7 (95% CI, 1.2-2.4). Prevalence of GUD was increased among case subjects, at visits 2 (OR, 3.2; 95% CI, 1.9-5.3) and 3 (OR, 2.1; 95% CI, 1.1-3.9). HIV load was increased in HSV-2-seropositive case subjects, compared with that in HSV-2-seronegative subjects, at 5 (P=.04) and 15 (P=.02) months after seroconversion. HIV acquisition is associated with HSV-2 seropositivity, and GUD is increased after seroconversion. HIV load is increased in HSV-2-positive subjects who seroconverted, suggesting a role for treatment of HSV-2 infection in HSV-2-seropositive, dually infected individuals.