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To investigate genital tract sources of HIV-1, we conducted extensive genitourinary sampling of 23 seropositive men without urethritis who shed HIV in their seminal plasma.Semen was collected, then samples were obtained for HIV RNA in blood plasma, urethral fluid, pre-prostate massage fluid/urine (PMF/U) and post-PMF/U, and expressed prostatic secretions. Systematic transrectal ultrasound-guided prostate biopsies obtained from multiple prostate areas were evaluated for HIV RNA and DNA.Seminal HIV RNA levels correlated with HIV RNA levels in urethral fluid and post-PMF/U and with prostate biopsies HIV DNA, but not with expressed prostatic secretions HIV RNA. However, only the HIV RNA level in post-PMF/U independently predicted that in semen (2.77-fold change in semen for each 10-fold change in post-PMF/U; 95% confidence interval, 1.0-7.7) accounting for one third of the seminal HIV RNA level variation, irrespective of adjustment for antiretroviral therapy.These data indicate that distal genitourinary sources other than the prostate appear to be the major source of seminal HIV in men without clinical urethritis or prostatitis. Because the HIV RNA level in blood plasma is not reliable as an independent clinical predictor of virus levels in seminal plasma, these findings also extend the concept that the male genital tract is a distinct virological compartment from blood.