The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca2+ compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca2+-ATPase activity was found to be responsible for intracellular Ca2+ transport in these cells, with no evidence of a mitochondrial Ca2+ transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A,, and stimulated by Na+ ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca2+ and H+ transport in promastigotes of P. francai.5215560