A preliminary screening of 511 persons at risk for AIDS living in southeastern Italy disclosed 20 cases of seroreactivity to human T lymphotropic viruses (HTLV). To verify and type the HTLV infection among these subjects, confirmatory serologic tests, polymerase chain reaction (PCR), and virus culture were done. No evidence of HTLV-I infection was found. HTLV-II infection was confirmed in 8 cases by HTLV-specific, synthetic peptide EIAs and PCR on uncultured cells; restriction analysis of the PCR-amplified env regions revealed the presence of HTLV-II/b strains in all 8 cases. Four sera were nontypeable by EIA. The finding of such indeterminate reactivities in a geographic area in which HTLV variants were previously described indicates the need for more extensive surveys among the healthy population. HTLV-II was isolated in 5 cases, and virus isolation was mostly dependent on the presence of an actively replicating human immunodeficiency virus type 1 in culture.