Six trypanosomatids isolated from different geographical areas from South America (Peru and Brazil) and different vectors and reservoir hosts (the triatomine Panstrongylus chinai [TP1], Triatoma infestans [TP2], Rhodnius ecuadorensis [TP3], R. prolixus [TB1], Didelphys marsupialis [TB2]), and one from a human asymtomatic patient [TB3], were characterized using lectin agglutination, isoenzyme profile, in vitro culture final metabolite patterns, and compared with a reference strain (Trypanosoma cruzi, Maracay strain [TC]). The different isolates were cultured in vitro in Grace's medium supplemented with 10% inactivated bovine foetal serum. According to our results and the statistical study, the isolate obtained from R. ecuadorensis should be designed as a Trypanosoma rangeli sp., showing all other isolates strong similarities to T. cruzi. Between them, two clusters could be identified, strongly correlating with the geographical origin. Cluster I grouped isolates from Peru and T. cruzi reference strain, and cluster II grouped the three Brazilian isolates. © 2003 Elsevier Science (USA). All rights reserved.