Four-color cell surface immunofluorescence and flow cytometry analysis was used to quantitate mononuclear cell subpopulations from HIV seropositive (HIV+) and seronegative (HIV-) homosexual men and heterosexual men. HIV+ men were divided into two groups based on peripheral blood CD4/mm3 of greater than 500 or less than 500. CD4+ cells that were simultaneously CD45R-, CDw29-, and 13- were significantly less in HIV+ men with less than 500 CD4/mm3 (17%) compared to heterosexual men (34%). This lower percentage of "CD4 only" cells in HIV+ males with less than 500 CD4/mm3 correlated with a significantly higher percentage of CD4+ cells that were CD45R+, CDw29+, and 13+ in these individuals. CD8+ cells that were CD45R+, 13+, but CD38-, were significantly less in HIV+ men with less than 500 CD4 as compared to HIV- homosexual men. In contrast, a second CD8+ subpopulation that was CD45R-, CD38+, and either 13+ or 13- was significantly greater in less than 500 HIV+ men as compared to both HIV- homosexual men and heterosexual men. A significant difference in this subpopulation was observed between the less than 500 and greater than 500 HIV+ groups and correlated with seropositivity for viral p24 antigen. Interestingly, CD8+ cells that were CD45R+, as well as CD38+, and either 13+ or 13- were significantly greater in the less than 500 HIV+ group compared to the greater than 500 HIV+ group, and did not correlate with p24 seropositivity. The percentage of monocyte/macrophages that were CD4- or expressed dim CD4 immunofluorescence, but were 13+, was significantly greater in HIV+ men (43%) compared to HIV- homosexual men (27%). In summary, we have identified previously undescribed mononuclear cell subpopulations that were altered with HIV infection and, in some cases, correlated with the stage of disease.