We have previously isolated and characterized a Trypanosoma cruzi cDNA encoding a polypeptide with a molecular mass of 52 kDa (Tc52) sharing significant homology to glutathione S-transferase. In the present study, by molecular and immunological approaches, we showed that Tc52 is preferentially expressed by dividing forms of the parasite: (e.g., epimatigotes and amastigotes). Moreover, we could identify the reactive antigen in different T. cruzi strains. A different pattern of reactivity on immunoblots was observed in the case of Trypanosoma rangeli. Furthermore, immunofluorescence assays using T. cruzi epimastigote culture forms revealed that the reactive antigen is localized within cytoplasmic organelles morphologically ressembling the structures previously designated as the reservosome found mostly at the posterior end of the parasite. Furthermore, the antibodies did not react against trypomastigotes which emerged from infected fibroblasts, whereas amastigotes showed polar fluorescence. Immunogold labeling and electron micrographs further revealed that the Tc52 protein is mainly associated with organelles composed of a large network of multivesicular structures, the latter being more abundant in epimastigotes. Taken together, these results demonstrated that Tc52 is associated with organelles composed of a multivesicular network and appears to be developmentally regulated, being fully expressed by parasite dividing forms.