No ideal test exists for Chagas' disease, and better diagnostic strategies are needed. We determined the diagnostic utility of an 85-kDa Trypanosoma cruzi protein in a multiple antigen binding assay (MABA). A standardized MABA test based on concentrated trypomastigote excretory-secretory antigen (TESA) and an 85-kDa purified protein showed 100% sensitivity and specificity. In field conditions, 6/66 individuals tested in a region not thought to be endemic (Rio Brito) were identified as seropositive for T. cruzi infection with our MABA test. In parallel, an enzyme-linked immunosorbent assay based on fixed epimastigotes detected 7/66 positives, which were independently confirmed. These data suggest that the 85-kDa and TESA proteins could be used in the MABA format as a complementary tool for the diagnosis of latent Chagas' disease. High anti-T. cruzi antibody detection rates, poor knowledge of Chagas' disease and its vector, and the demonstration of infected vectors in the study community all suggest a significant risk of reemergence of T. cruzi infection in this region of Venezuela.