Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain most of the cellular calcium. The data presented here demonstrate that Leishmania donovani acidocalcisomes possess a Na+/H+ exchanger. 3,5-Dibutyl-4-hydroxytoluene, in the concentration range of 0–20 μM, inhibited the Na+/H+ exchanger, and strongly stimulated the activity of the vacuolar H+-ATPase responsible for vacuolar acidification. As occurs with Na+, the cationic anti-leishmanial drugs pentamidine, WR-6026, and chloroquine promoted a fast and extensive alkalization of the L. donovani acidocalcisomes.