When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7 microM. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor alpha-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500 microM vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.