It is well established that HIV is found in semen, either as cell-free or cell associated virus, yet many questions remain about the source of the virus. A number of factors, including anatomic features of the male reproductive tract, the restricted access of the immune system to the germ cell compartment, and the results from sexually transmitted virus studies, suggest that the source of HIV in semen may be different from that in the peripheral blood. In this study, we examine the HIV in the infected cells of semen as indicators of the virus producing reservoir. The frequency of HIV positive leukocytes in semen is compared to that of concurrent blood samples from eight donors and these values are found to be highly variable and frequently discordant. The protease gene sequences of HIV strains isolated from semen cells and blood cells were determined and phylogenetic analyses were performed which indicate the virus populations in the two sources are genetically distinct. In one patient receiving anti-HIV protease inhibitor therapy, gene sequences indicative of protease inhibitor resistance were found in the blood, but not the semen cell compartment. These results suggest that HIV in the semen and blood compartments are distinct, and further, may respond differently to antiviral therapy.