Areas where mucocutaneous leishmaniasis, kala-azar and Chagas' disease are prevalent often overlap in Latin America. Due to a variable degree of cross-reactivity between the three etiological agents and antibodies synthesized during the course of the disease, a test able to distinguish between them is greatly needed. Saline-extracted antigen from live Trypanosoma cruzi epimastigotes behaved towards Chagas' disease sera in immunoenzymatic assays (ELISA) with the same specificity and sensitivity as the antigen used routinely for such tests. In tests using mucocutaneous leishmaniasis or kala-azar sera, the live T. cruzi antigen showed a much lower geometric mean titer (GMT) than the standard T. cruzi antigen. In ELISA tests with L. braziliensis antigen a higher GMT was seen with kala-azar sera than with those from cases of mucocutaneous leishmaniasis. Also, sera from Vale do Ribeira leishmaniasis showed higher GMT with an L. donovani antigen than with a homologous one. Such discrepancies were not seen in immunofluorescence tests employing the same sera and antigens.