Peripheral blood leukocytes (PBL) from male homosexual and heterosexual volunteers who did not have evidence of acquired immunodeficiency syndrome (AIDS) were studied for their ability to generate cytotoxic T lymphocyte (CTL) responses in vitro to allogeneic stimulator PBL from a single individual or from a pool of donors (allo-pool). Seventeen of 39 homosexual donors generated strong primary CTL activity to a single randomly selected stimulator donor, whereas only two of 16 heterosexual donors generated a strong CTL response to the same stimulators. A more detailed study was performed by using PBL from 11 of the homosexual and five of the heterosexual donors, in which CTL responses to the allo-pool were repeatedly tested over a 14-mo period. The response status of the donors that were strong and weak or moderate responders did not change during this period. No correlation of strength of CTL activity was observed with the following: HLA mismatching between responder and stimulator; proportion of OKT4+ and OKT8+ subsets in peripheral blood; antibodies to HTLV-III; antibody titers to hepatitis B, Epstein Barr virus, cytomegalovirus, or HLA alloantigens; homosexual practices; or circulating immune complex levels, although a statistical correlation by multivariate analysis was observed between elevated allogeneic CTL and a combination of other factors. The findings are discussed with respect to possible relevance to AIDS susceptibility and development.