To investigate serological, epidemiological, and molecular aspects of HTLV-1, HTLV-2, and HIV-1 infections in Amerindian populations in Brazil, we tested 683 and 321 sera from Tiriyo and Waiampi Indians, respectively. Both HIV-1 and HTLV-2 infections were detected at low prevalence among the Tiriyos whereas only HTLV-1 was present among the Waiampis, also at low prevalence. Analysis of the nucleotide sequence of the 631 bp of the env gene obtained from the three HTLV-2 isolates detected among the Tiriyos demonstrated by restriction fragment length polymorphism that these viruses belong to subtype IIa. Phylogenetic analysis of this same fragment showed that these sequences cluster closer to HTLV-2 isolates from intravenous drug users living in urban areas of southern Brazil than to the same gene sequence studied in another Brazilian tribe, the Kayapos. Our results confirm the distribution of Brazilian HTLV-2 sequences in a unique cluster I and cluster IIa and suggest that there is a considerable degree of diversity within this cluster. We also report for the first time HIV-1 infection among Brazilian Amerindians.