The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5-mu-M Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1-mu-M FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500-mu-M vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8-mu-M. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [H-3]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.522251262