Following purification by affinity chromatography, three glutathione-binding proteins (TcGBP) of 45, 30, and 25 kDa were co-purified from Trypanosoma cruzi epimastigotes. Using 1-chloro-2,4 dinitrobenzene as substrate, a glutathione S-transferase activity of 70 nmol/min/mg of proteins was detected in the GSH binding fraction. An increased expression of TcGBP and total GST activity was observed upon incubation of parasites with phenobarbital, which is an inducer of GST synthesis. Immunofluorescence and electron microscopic experiments demonstrated that TcGBP were expressed by all developmental stages of the parasite, including infective forms. The expression of these proteins by intracellular dividing amastigotes could be in favour of a potential defensive role of these molecules against host attack. Results obtained by immunoprecipitation of in vitro translation products using anti-TcGBP antisera suggested that these three polypeptides are not glycosylated. In addition, antibodies directed against the TcGBP were found in a high proportion of T. cruzi-infected chronic chagasic patients' sera and in sera of chronically infected BALB/c mice. In contrast, acute chagasic patients' sera and acute-phase mouse sera were found to be poorly reactive with these proteins. Our results identify a new class of potential target antigens, which may be essential for the development of T. cruzi in its host. Their protective role in experimental models deserves to be investigated.