The prevalence of Chagas' disease in Ayoreo amerindians living in their traditional habitat of the Paraguayan Chaco was assessed by ELISA and indirect immunofluorescence techniques. The Ayoreo is the last hunther-gatherer tribe of the Paraguayan Chaco and was "pacified" between 1965 and 1975. They still maintain a seminomadic way of life. Chagas' disease prevalence in the Ayoreo communities probably represents their natural prevalence in Chaco Amerindians before settlement. Triatoma sordida was identified as the insect vector in the settlements of Gesudi and Chovoreca, while the highly domiciliary Triatoma infestans was found in Campo Loro. The prevalence of Chagas' disease in Gesudi and Chovoreca, based on our serological surveys was 12-13%. Other authors reported a 72.9% of Chagas' disease prevalence in amerindians settled in the Chaco since 1930-1940.