Highly purified Trypanosoma-decay accelerating factor (T-DAF), a 87–93-kD glycoprotein present on the surface of metacyclic and trypomastigote forms of Trypanosoma cruzi, was used as antigen to evaluate the presence of specific serum antibodies in experimentally infected mice and patients with Chagas' disease by enzyme-linked immunosorbent assay (ELISA). Mouse T-DAF antibodies were first recorded on day 7 postinfection, reached maximal concentrations on day 30, and maintained at positive titers thereafter. High immunogenicity was clearly demonstrated by the detection of T-DAF antibodies in 96% of the sera collected from chagasic patients in either the acute or the chronic phase of the disease. Control sera from normal individuals and from patients with leishmaniasis or other chronic infections did not give positive results. Serologic evaluation using T-DAF as antigen did not discriminate between patients with the cardiac and the digestive forms of the disease. The performance of the T-DAF ELISA was compared with that of conventional screening tests for Chagas' disease (indirect immunofluorescence and hemagglutination). The T-DAF ELISA test showed a sensitivity of 96%, a specificity of 100%, an efficiency of 99%, a positive predicted value of 100%, a negative predicted value of 98%, and a kappa index of 0.96, thus indicating that it can be successfully used for the serodiagnosis of T. cruzi infection in humans.