We investigated the presence of human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) infections, first searching for specific antibodies in 553 serum samples obtained from HIV-1-infected patients from São Paulo, Brazil. Sera were screened using two enzyme-linked immunosorbent assays (ELISAs): the ELISA-EM (ELISA HTLV-I/II, EMBRABIO, BR), which contains HTLV-I and HTLV-II lysates, and the ELISA-DB [ELISA HTLV-I/II, Diagnostic Biotechnology (DB), Singapore], which contains HTLV-I lysate, and HTLV-I and HTLV-II recombinant env proteins (MTA-1 and K55, respectively). Serum samples showing two positive and/or borderline results were confirmed by Western blot (WB 2.3, DB), which discriminates HTLV-I from HTLV-II. WB analyses disclosed 22 cases (4.0%) of HTLV-I and 34 (6.1%) of HTLV-II seroreactivity; 24 sera had indeterminate antibody profile (4.3%) and 2 specimens showed reactivity to both MTA-1 and K55 env proteins. Using stringent WB criteria and analyzing the population according to risk factors, the prevalence rates of HTLV-I and HTLV-II infections were 11.2% and 16.8% in i.v. drug users, 3.4% and 5.5% in heterosexual individuals, and 1.4% and 2.2% in homosexual/bisexual men, respectively. A comparison of ELISA and WB results disclosed that both ELISAs were highly sensitive in detecting HTLV-I antibodies, whereas the ELISA-DB showed 82% sensitivity and the ELISA-EM 100% sensitivity in detecting HTLV-II antibodies. PCR analyses conducted on 37 representative cells samples confirmed the presence of HTLV proviral DNA in the majority of concordant serological cases, except in one, which was HTLV-I infected and seroreacted with K55 protein of HTLV-II. Indeed, after PCR, one case of HTLV-I infection and HTLV-II coinfection, and 30% of WB-seroindeterminate or inconclusive cases infected with HTLV-II could be detected. Our data stress high prevalences of both HTLV-I and HTLV-II infections in HIV-1 coinfected i.v. drug users from São Paulo, and suggests that ELISA kits containing only K55 protein as the HTLV-II-specific antigen, may not have the appropriate sensitivity for the detection of HTLV-II infection in this geographic region, pointing out the need of improved screening tests to be used in Brazil.