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Herpes simplex virus type 2 (HSV-2) infection is associated with an increased risk for acquiring HIV, but little is known about the temporal sequence of these infections.Six thousand three hundred ninety-six men were evaluated for serologic HSV-2 and HIV infections and behaviors during a male circumcision trial in Rakai, Uganda.HIV and HSV-2 status were determined using enzyme-linked immunosorbent assays and confirmed by HIV-1 and HSV-2 western blots. A Poisson multivariable model was used to estimate adjusted incidence rate ratios of HIV acquisition associated with HSV-2 and other covariates.HIV incidence was 1.09/100 person-years and acquisition was associated with incident HSV-2 infection [adjusted incidence rate ratio (adjIRR) 5.28, 95% confidence interval (CI) 2.79–9.98], chronic HSV-2 infection (adjIRR 2.78, 95% CI 1.64–5.68), genital ulcer disease, urethral discharge, genital washing after intercourse, being unmarried, and being uncircumcised. Sixteen men acquired both HIV and HSV-2 during the trial: four acquired HIV first, three acquired HSV-2 first, and nine acquired both infections in the same follow-up interval.The findings suggest that unsafe sex places men at risk of both HIV and HSV-2 infections, and it is unclear whether HSV-2 acquisition is a cofactor for HIV infection or a marker of correlated sexual exposures. This reinforces the need for promotion of safe sex as the primary method of prevention of both viruses.