Semen is the body fluid most commonly associated with sexual transmission of human immunodeficiency virus type-1 (HIV-1). Because the male genitourinary tract is distinct immunologically from blood, compartment-dependent factors may determine HIV-1 shedding in semen. To identify these factors, the authors obtained 411 semen and blood specimens from 149 men seen up to three times. Seminal plasma was assayed for HIV-1 RNA and semen was cocultured for HIV-1 and cytomegalovirus (CMV), which may up-regulate HIV-1 replication. The best multivariate model for predicting a positive semen HIV-1 coculture included two local urogenital factors, increased seminal polymorphonuclear cell count (odds ratio (OR) = 12.6 for each log10 increase/mL, 95% confidence interval (CI) 12.2, 134.5) and a positive CMV coculture (OR = 3.0, 95% CI 1.2, 7.7). The best multivariate model for predicting semen HIV-1 RNA included two systemic host factors, CD4+ cell counts <200/microliter (OR = 3.0, 95 percent CI 1.3, 6.9) and nucleoside antiretroviral therapy (monotherapy: OR = 0.5, 95% CI 0.3, 1.0; combination therapy: OR = 0.4, 95% CI 0.2, 0.9), and a positive CMV coculture (OR = 1.7, 95% CI 1.0, 3.0). Thus, both systemic and local genitourinary tract factors influence the risk of semen HIV-1 shedding. These findings suggest that measures of systemic virus burden alone may not predict semen infectivity reliably.