The relative specificities and sensitivities of several serological assays for the diagnosis of Trypanosoma cruzi infection were estimated in Indian populations of Argentina and Paraguay. The results obtained with the assays, which proved to be most reliable, were used to study the distribution of the parasite in these populations. Serological evidence of T. cruzi infection was demonstrated in 256 (37.7%) of 679 Indians living in relatively small and isolated communities in the Salta province of northern Argentina and in western Paraguay, regions that are part of the tropical territory called Gran Chaco. In contrast, none of the 94 Indians examined in south-western Argentina was positive. Infection in the Gran Chaco Indians increased with age and clustered in families. Marked differences in seroprevalence were observed between the 16 Indian communities examined in Gran Chaco. These differences seem to be associated both with the risk of transmission from the sylvatic reservoirs of the parasite and with the frequency with which vector-spraying campaigns have been implemented.