Healthy homosexual men between the ages of 21 and 65 years, from the Washington, DC (n=162), and New York City (n=89) areas, were studied for antibodies in the serum against cytomegalovirus (CMV), herpes simplex virus (HSV) types 1 and 2, and Epstein Barr virus (EBV) viral capsid antigen (VCA). CMV-specific antibodies were assayed by enzyme-linked immunosorbent assay (ELISA), anti- HSV-1 and HSV-2 antibodies were measured by indirect hemagglutination (IHA), and antibodies to EBV VCA were measured by the immunofluorescence assay. Antibodies to human T lymphotrophic virus III (HTLV-III) were detected by ELISA and Western blot procedures. T lymphocytes were enumerated using OKT4 monoclonal antibody. Healthy male volunteer blood donors (n=90) matched for age range and race proportions were used as controls. The percentage of seropositive individuals in the homosexual group was higher (90-98%) for all the viruses tested than in the control group (47-87%). Comparisons of the geometric mean titers, expressed as reciprocal serum dilutions, of seropositive individuals in homosexual (H) vs control (C) group were as follows: CMV-IgG (ELISA) H = 1:794, C = 1:68; HSV-1 (IHA) H = 1:248, C = 1:14; HSV-2 (IHA) H = 1:56, C = 1:17; EBV- VCA (IFA) H = 1:131. The homosexual group also showed a higher frequently of individuals with elevated titers than the control group. The CMV IgM antibody was prevalent in 17.7% of the homosexual group and 5% of the control group; arithmetic means for ELISA values for CMV IgM were 0.207 for the homosexual group and 0.05 for the control group. In the homosexual group, the anti-CMV antibody titers increased with age (P=0.01) and with numbers of sex partners (P=0.06). Both anti-HSV-1 and anti-HSV-2 antibodies correlated with the number of sex partners (P-0.04 and P=0.05, respectively). Neither age nor partner number correlated with response to EBV, and no particular sex act was related to the EBV VCA titer level.