In the present report we describe the use of Trypomastigote Excreted-Secreted Antigens (TESA) as antigen in ELISA for Chagas' disease serodiagnosis. The study was carried out on 284 patients, 164 of whom were nonchagasic subjects including individuals with leishmaniasis or other pathologies, and 120 chagasic patients, being 53 in the acute (with positive IgA and IgM antibodies to T. cruzi) and 67 in the chronic phase. TESA-ELISA showed 100% positivity in the survey of IgG antibodies in chagasic patients (acute and chronic) and 100% positivity for IgM antibodies in acute phase sera. TESA preparation does not require biochemical purification procedures and does not present the cross-reactivity of leishmaniasis sera observed when ELISA with epimastigote alkaline extract (EAE) is performed. ELISA competition assays showed that anti-T. cruzi antibodies of sera from chagasic patients that react with TESA are different from those that react with EAE. Besides, partial characterization of TESA showed that several epitopes present in this fraction are absent in EAE.