Serologic evidence of Trypanosoma cruzi infection was demonstrated in 43.5% of 519 Paleoamerindians and in only 2.5% of 161 non-Indians (Mennonites of German descent and Paraguayans of Spanish descent) inhabiting an area of western Paraguay that belongs to the Gran Chaco territory. These people ranged in age between two and 80 years. All were also tested for infection with the human T cell lymphotropic virus type II (HTLV-II). The prevalence of HTLV-II infection was 22.1% in Indians and 3.7% in non-Indians. As determined by a multivariate logistic regression analysis that controlled for relevant confounders, an HTLV-II-infected individual was 2.28 times more likely to be seropositive for T. cruzi than an HTLV-II negative. Possible explanations for this finding are discussed. The difference in T. cruzi prevalence between Indians and non-Indians was associated with differences between these groups in exposure to known risk factors for infection with the parasite. There were significant differences in the seroprevalence of T. cruzi among the two predominant Indian groups, even when they inhabited communities that were close to each other. These differences were associated with differences in the prevalence of HTLV-II infection but not with differences in exposure to known risk factors for T. cruzi infection. Infection with T. cruzi increased with age, was greater in males than in females, and clustered in families.