We have demonstrated that the polymerase chain reaction is a valid and sensitive technique for the detection of human immunodeficiency virus type 1 (HIV-1) proviral deoxyribonucleic acid (DNA) in human semen. The combination of extraction, polymerase chain reaction, and liquid hybridization techniques used in this study was sensitive to a level of detection of one HIV-1 infected cell in 100,000 (or 3 infected cells/test sample). In a series of matched peripheral blood mononuclear cells (PBMC) and semen cells from 25 HIV-1 seropositive homosexual men, HIV-1 DNA was detected by polymerase chain reaction in 23 of 25 PBMC samples and 1 of 25 semen samples. By coculture on mitogen-activated peripheral blood leukocyte target cells, 19 of 24 PBMC and 4 of 24 semen samples were positive for infectious HIV-1. Of the four culture-positive semen samples, three were negative for the proviral form of the virus in the polymerase chain reaction assay. These data indicate that HIV-1 infected cells are not as prevalent in semen as in the peripheral blood. Furthermore, they indicate that the classical polymerase chain reaction approach, which only detects HIV-1 proviral DNA (infected cells), is not sufficient for clinical screening programs whose goal is the detection of HIV-1-infected semen samples. Accurate semen analysis by polymerase chain reaction may require enrichment of the infected cell population and/or a reverse transcriptase step to enable detection of the infectious ribonucleic acid form of the virus.