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This study was conducted to compare viral dynamics in blood and semen between subjects with antibody negative, acute HIV-1 infection and other subjects with later stages of infection.A prospective cohort study was embedded within a cross-sectional study of HIV screening in a Lilongwe, Malawi STD clinic.Blood samples from HIV antibody negative or indeterminate volunteers were used to detect HIV RNA in plasma using a pooling strategy. Blood and seminal plasma HIV-1 RNA concentrations were measured over 16 weeks.Sixteen men with acute HIV infection and 25 men with chronic HIV infection were studied. Blood viral load in subjects with acute HIV infection was highest about 17 days after infection (mean ± SE, 6.9 ± 0.5 log10 copies/ml), while semen viral load peaked about 30 days after infection (4.5 ± 0.4 log10 copies/ml). Semen viral load declined by 1.7 log10 to a nadir by week 10 of HIV infection. Semen and blood viral loads were more stable in chronically infected subjects over 16 weeks. Higher semen levels of HIV RNA were noted in subjects with low CD4 cell counts.These results provide a biological explanation for reported increases in HIV transmission during the very early (acute) and late stages of infection. Recognizing temporal differences in HIV shedding in the genital tract is important in the development of effective HIV prevention strategies.