Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope-tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.