High levels of human immunodeficiency virus (HIV) in rectal secretions and semen likely increase the risk of HIV transmission. HIV-infected men who have sex with men made 2-3 study visits, over 4 weeks, to assess rectal, seminal, and plasma levels of HIV RNA. Mixed-effects models estimated the effect of factors on HIV shedding. Twenty-seven (42%) of 64 men were receiving antiretroviral therapy (ART); regardless of ART use, median HIV RNA levels were higher in rectal secretions (4.96 log10 copies/mL) than in blood plasma (4.24 log10 copies/mL) or seminal plasma (3.55 log10 copies/mL; P < .05, each comparison). ART was associated with a 1.3-log10 reduction in rectal HIV RNA in a model without plasma HIV RNA; with and without plasma RNA in models, ART accounted for a >1-log10 decrease in seminal HIV RNA levels. Thus, controlling for plasma HIV RNA, ART had an independent effect on seminal, but not rectal, HIV levels.