To assess the prevalence of HTLV-I and HTLV-II infections in different groups at risk for HIV-I infection, a study on 867 subjects was carried out by means of serological and PCR analyses. Serum specimens were collected from 268 intravenous drug users (IVDU), 66 homosexual men, 248 subjects with sexually transmitted diseases (STD), 105 thalassemics and 180 hemophiliacs. Sera from 3 IVDU and a sample from an STD patient were confirmed as HTLV-II seropositive; a thalassemic patient was seropositive for HTLV-I; a homosexual man, though confirmed as HTLV-I/II-seroreactive, could not be typed by serological methods. No hemophiliac was found to be HTLV-I/II-reactive. All 3 HTLV-II-seroreactive IVDU and the HTLV-I-infected thalassemic were confirmed by PCR; an additional sample from an IVDU, indeterminate by Western blot, was confirmed to be positive for HTLV-II by PCR. Subtyping of HTLV-II samples indicated the presence of II/b subtype in all 4 cases. Up to now, the reservoir for HTLV-II infection in southeastern Italy is mainly represented by IVDU, while HTLV-I infection seems to be sporadic.