The prevalence of antibodies to HTLV-I in 5,475 Portuguese from 6 regions spanning the country was studied. Overall seroprevalence was 0.55%, indicating that Portugal is not an endemic area for this virus. Seropositives were distributed throughout the country, and no geographic clustering was observed. The seroprevalence of individuals who had lived in former Portuguese colonies in Africa (0.7%) was significantly higher than that of individuals who had not been in Africa (0.36%). An increase in seroprevalence with age was noted, and more females than males were antibody-positive, though not significantly so. Serum from one donor (1711), originating from Guinea-Bissau, was shown by Western blot and radioimmune precipitation to react with various proteins of HTLV-I, HIV-1 and -2, and SIV. Based on the serologic profiles and isolation of bona fide HTLV-I from her lymphocytes (confirmed by immunologic analysis, molecular cloning of the provirus and restriction enzyme analysis and sequencing of the DNA), together with the reactivity of her sera with an HIV-2 isolate obtained from her husband, we conclude that this donor was doubly infected with HTLV-I and HIV-2, rather than being the host to an as yet unidentified retrovirus.