Antigens of the epimastigote stage of Trypanosoma cruzi were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and examined for their ability to bind antibodies in sera from humans infected with this organism or infected with one or both of the related protozoa Leishmania braziliensis and Leishmania donovani by protein blot analysis and enzyme-linked immunosorbent assay. Most of the antigens were bound by antibodies against each one of the organisms. A group of antigens with Mrs between 31,000 and 21,000 were bound by antibodies against T. cruzi only. These antigens were isolated and used in an enzyme-linked immunosorbent assay for the differential diagnosis of Chagas' disease, with excellent results. All sera from individuals proven to be infected with T. cruzi reacted with the antigens, whereas none of the sera from individuals proven to be infected with L. braziliensis or L. donovani reacted with the antigens, even when tested at a low dilution. Biochemical characterization of the isolated antigens revealed the presence of protein and carbohydrate. The reactivity of the isolated antigens with antibodies was completely abolished by pronase and partially abolished by sodium periodate. Protein blot analysis of sera from mice immunized with the antigens revealed a major large band with an Mr between 31,000 and 21,000 and a minor band with an Mr of 45,000, suggesting sharing of epitopes between antigens of different Mrs. These sera did not agglutinate or lyse live epimastigotes. Indirect immunofluorescent antibody tests with live and dead epimastigotes revealed that antibodies in the sera only bound to Formalin-killed organisms.