In this work, we show the kinetics of pyrophosphate-driven H+ uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K+ and PPi. H+ was released with the addition of Ca2+, suggesting the presence of a Ca2+/H+ antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H+-pyrophosphatase with characteristics of type I V-H+-PPase. However, we did not find any evidence, either for the presence of H+-ATPases or for Na+/H+ exchangers in these acidocalcisomes.