Contact with semen of seropositive men is important for sexual transmission both human immunodeficiency virus (HIV) type 1 and human cytomegalovirus (CMV), but the factors that determine shedding of either virus in semen are poorly understood. HIV was cultured from 36 (17%) of 215 semen specimens from 56 seropositive men, and CMV was cultured from 42 (30%) of 139 specimens. In logistic regression models, the CD8+ cell count in peripheral blood was the best predictor of HIV shedding in semen. Shedding of HIV was more closely associated with concomitant shedding of CMV than with CD4+ cell count, and antiretroviral therapy had minimal influence on shedding of HIV. In contrast, CD4+ cell count was the best predictor of CMV shedding in semen. Factors that determine shedding of viruses in semen may differ substantially from those that influence virus levels in the systemic immune compartment. Likewise, immunologic factors that determine shedding of HIV appear to differ from those that control shedding of CMV in semen.