Trypanosoma cruzi expresses oligopeptidase B and cathepsin B that have important functions in the interaction with mammalian host cells. In this study, we demonstrated that sera from both chagasic rabbits and humans have specific antibodies to highly purified native oligopeptidase B and cathepsin B. Levels of antibodies to cathepsin B were higher than those observed to oligopeptidase B by absorbance values recorded upon ELISA. We next showed that 90% and 30% of sera from individuals with mucocutaneous leishmaniasis have antibodies that recognize oligopeptidase B and cathepsin B as antigens, respectively. In addition, 55% and 40% of sera from kala-azar patients have antibodies to oligopeptidase B and cathepsin B, respectively. Sera from malaria patients did not recognize the proteases as antigens. Despite high levels of specific antibodies, sera from T. cruzi-infected patients did not inhibit the activities of either oligopeptidase B or cathepsin B. Furthermore, sera or IgG purified from either infected or non-infected individuals enhanced the enzymatic activity of the secreted oligopeptidase B. Oligopeptidase B secreted by trypomastigotes and cathepsin B released upon parasite lysis retain their enzymatic activities and may be associated with Chagas' disease pathogenesis by hydrolyzing host proteins and inducing host immune responses.