The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the 'acidocalcisome' and which appears to be a unique organelle present in trypanosomatids.