We describe for the first time the presence of human T lymphotropic virus type II (HTLV-II) infection in Venezuela, among the Pume Amerindians living in the southern plains of the country. Antibodies to HTLV-II antigens were assessed by enzyme immunoassays (Elisa), Western blot, radioimmuno-precipitation, and immunofluorescence; titration studies against HTLV-I- and HTLV-II-infected cell lines were very useful in the differentiation of HTLV-I and HTLV-II antibodies. The HTLV-II general prevalence was 5%; however, there is a striking difference in prevalence between the truly isolated villages (0%) when compared to those living along the riverside and thus in contact with outsiders (9%). Preliminary evidence suggests sexual contact as the main source of transmission. These findings might suggest that HTLV-II in Venezuela originated through contact with outsiders rather than ancient infection related to the origins of the Pume.