To estimate the presence of, and the risk factors for HTLV-I and HTLV-II infections among HIV-1 infected subjects in Sao Paulo, Brazil, a serosurvey was performed in 471 HIV-1 infected patients, including 216 intravenous drug addicts (IVDA), 229 homosexual/bisexual men, and 26 with other risk factors. Serum samples were screened for HTLV seroreactivity by ELISA; reactive samples were analyzed by Western Blot (WB), using whole HTLV-I lysate as antigen. To confirm and discriminate HTLV-I and HTLV-II infections, sera presenting any bands on WB were further analyzed by a WB containing recombinant HTLV-I and HTLV-II proteins (WB 2.3), and by enzyme immunoassays using synthetic peptides specific for envelope proteins (Synth-EIA). In 22 cases, cell samples were available for polymerase chain reaction (PCR) studies. On WB, 114 sera were reactive and, of these, 37 and 25 were concordantly positive on both WB 2.3 and Synth-EIA procedures for HTLV-I and HTLV-II specific antibodies, respectively; 37 specimens were negative on both assays, and 15 gave discordant or indeterminate results. PCR findings confirmed concordant results obtained in the discriminatory serological assays. The prevalence rates of HTLV-I and HTLV-II infections were 15.3% and 11.1% in IVDA, and 0.9% and 0.4% in homosexual/bisexual men, respectively. No case of HTLV-I/HTLV-II co-infection was found. PIP: HTLV-I is associated with adult T-cell leukemia/lymphoma and a neurological disorder known as HTLV-I-associated myelopathy or tropical spastic paraparesis. HTLV-II was initially isolated from subjects with a T-cell variant of hairy cell leukemia, but its etiological role in that or other diseases is unclear. HTLV infections, like HIV, are transmitted sexually, via blood transfusion and contaminated needles, and from mother to infant. Many reports indicate that HTLVs are present in the same populations at risk for HIV-1, and the cofactorial role of HTLVs in AIDS progression has been suggested by in vitro studies and epidemiological data. The authors report findings from a serosurvey conducted among 216 HIV-seropositive male and female intravenous drug users (IVDU), 229 HIV-seropositive homosexual and bisexual men, and 7 HIV-seropositive men and women who had had multiple transfusions, and 19 HIV-seropositive heterosexual men with multiple partners to estimate the presence of and the risk factors for HTLV-I and HTLV-II infections among HIV-1 infected individuals in Sao Paulo, Brazil. 70.9% of the subjects were classified according to CDC criteria as having AIDS. ELISA, Western blot, and polymerase chain reaction methods were used. The prevalence rates of HTLV-I and HTLV-II infections were 15.3% and 11.1% in IVDUs, and 0.9% and 0.4% in homosexual and bisexual men, respectively. No case of HTLV-I/HTLV-II co-infection was observed.