We investigated acute human immunodeficiency virus (HIV) infection among men enrolled in a genital ulcer treatment trial in South Africa. HIV-negative participants were tested at baseline by HIV RNA polymerase chain reaction and followed up after 1 month to measure HIV seroconversion. There were 228 HIV-negative men at baseline; 10 were positive for HIV RNA, and 8 seroconverted to HIV at day 28. The prevalence of acute HIV among HIV-negative men at baseline was 18 (7.9%) of 228 men (95% confidence interval [CI], 4.4-11.4) and 18 (2.9%) of 615 men (95% CI, 1.6-4.3) in the overall study population. These data highlight the importance of genital ulcer patients in HIV transmission. Trial Registration. ClinicalTrials.gov identifier: NCT00164424 .