Three hundred seventy-nine individuals [137 non-injecting drug using (non-IDU) heterosexuals, 130 homosexual men and 112 IDU] attending the human immunodeficiency virus (HIV) testing program of a sexually transmitted disease (STD) clinic in Rome were studied to estimate the prevalence and to identify the modalities of transmission of human herpesvirus-8 (HHV-8) infection. Serological analysis was performed by using an immunofluorescence assay able to detect anti-latent and anti-lytic HHV-8 antibodies. Twelve acquired immunodeficiency syndrome (AIDS)-Kaposi's sarcoma (KS) patients and 94 blood donors were tested as reference population groups. Anti-lytic antibodies were detected in 185 (48.8%) individuals; 52 of them (13.7%) also had anti-latent antibodies. Both anti-lytic and anti-latent antibody prevalence were higher among homosexual men (66.9% and 27.7%, respectively) than among IDU (49.1% and 8.0%, respectively) and non-IDU heterosexuals (31.4% and 5.1%, respectively), and tended to increase with age. Anti-lytic HHV-8 antibodies were associated with syphilis [odds ratio (OR)=3.81] but not with hepatitis C virus (HCV) seropositivity. HIV-infected homosexual men were more likely to have HHV-8 antibodies than those who were HIV-negative. When using anti-latent antibodies the direction of the OR remained the same, although the associations did not often reach statistical significance. Among AIDS-KS patients, 83.3% had anti-lytic and 66.6% had anti-latent antibodies. Among blood donors, 28% had anti-lytic antibodies and 2 of them (2.1%) also had anti-latent antibodies. Our data indicate that HHV-8 seroprevalence increases with age and is higher among homosexual men, particularly those infected with HIV. This is consistent with sexual transmission of HHV-8 infection. In addition, the presence of HHV-8 antibodies in HIV-negative non-IDU heterosexual contacts and in healthy blood donors is consistent with the high incidence of classic KS in Italy.