Epidemiologic studies indicate that human T-cell lymphotropic virus type I (HTLV-I), the causative agent of most cases of adult T-cell leukemia/lymphoma (ATLL) in Southeast Japan and the Caribbean islands and the probable cause of a progressive neurological disorder often referred to as tropical spastic paraparesis, occurs with unusual geographic clustering. The current large-scale serosurvey was undertaken to improve our understanding of HTLV-I prevalence in different parts of the world. We analyzed 43,445 serum samples collected from various geographic locales worldwide; 76% of these sera came from clinically healthy donors. Samples were initially screened by an enzyme-linked immunosorbent assay (ELISA) and 4,353 were further evaluated by means of competition assays. In this study, which did not include sera from endemic areas of Japan, a high prevalence of infection was observed in several countries in the Caribbean basin. A significant age-sex difference was observed between populations in the Caribbean and non-endemic regions of Japan. The reason for the male excess in non-endemic areas of Japan will require further study, while the female excess in the Caribbean basin is compatible with the previously described pattern for other HTLV-I-endemic areas. A newly recognized area of possible endemicity was southern Florida, where evidence of infection with HTLV-I or a related virus was found in a group of native Americans whose sera were collected in 1968. In certain parts of the world, particularly sub-Saharan Africa, important problems in determining specificity of reactivity occurred, probably because of cross-reacting antibodies. No pattern was detected that could explain the cross-reactivity solely on the basis of geographic areas, specific patterns of non-viral parasitic infection, or methods of handling the specimens. It is possible that these cross-reactivities are antibodies to proteins from HTLV-I-related retroviruses yet to be discovered.