Antiretroviral therapy may lead to decreased shedding of human immunodeficiency virus type 1 (HIV-1) in genital secretions. Thirty men, 19 receiving amprenavir and 11 receiving amprenavir, zidovudine, and lamivudine, donated blood and semen while undergoing treatment, to evaluate the effects of these medications on HIV-1 shedding in semen. Before therapy, 4 men had HIV-1 RNA levels in seminal plasma >6.0 log10 (1 million) copies/mL, markedly higher than levels in blood plasma. Most men (77%) had HIV-1 RNA levels in seminal plasma below the limit of quantification during therapy. Amprenavir alone suppressed HIV-1 RNA levels to <400 copies/mL in seminal plasma in the majority of patients, the first direct demonstration of the antiretroviral effects of a protease inhibitor in the male genital tract. However, 8 men (27%) had measurable HIV-1 in seminal plasma at their last study visit, 4 with increasing levels. Persistent replication of HIV in the genital tract may have implications for the selection of resistant virus and sexual transmission of HIV-1.