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Six hundred intravenous drug users (IVDUs) and two hundred North Africans were screened for human T-cell leukemia virus (HTLV) antibodies using several serological methods. Eighteen of the eighty-two HTLV-seropositive individuals were also tested by the polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), a non-isotopic method of immunoenzymatic detection of the amplified DNA. Of these eighteen subjects, eight IVDUs were found to be HTLV-II-positive by the PCR-DEIA, whereas all of the eighteen subjects were negative for HTLV-I. Western blot (WB) confirmed six of the eight HTLV-positive subjects, while the results of the remaining two were indeterminate. The results confirmed the PCR-DEIA as a rapid and an efficient method of discriminating between HTLV-I and HTLV-II infection, whereas serological tests, including the WB, have limitations in terms of specificity and sensitivity. Moreover, this study showed a higher frequency of HTLV seroreactivity in the Italian IVDU population than in previous studies and confirmed that HTLV-II is more frequent than HTLV-I in this population.