Trypanosoma cruzi epimastigotes maintained an intracellular free calcium concentration of about 0.15-mu-M, as measured with the fluorescent indicator Fura-2. The maintenance of low [Ca2+]i is energy-dependent since it is disrupted by KCN and FCCP. When the cells were permeabilized with digitonin, the steady-state free Ca2+ concentration in the absence of ATP was about 0.7-mu-M. The additional presence of ATP resulted in a steady-state level close to 0.1-0.2-mu-M which compares favorably with the concentration detected in intact cells. Intracellular Ca2+ uptake at high levels of free Ca2+(> 1-mu-M) was due to energy-dependent mitochondrial uptake as indicated by its FCCP-sensitivity. However, as the free Ca2+ concentration was lowered from 1-mu-M, essentially all uptake was due to the ATP-dependent Ca2+ sequestration by the endoplasmic reticulum as indicated by its stimulation by ATP, and its inhibition by sodium vanadate. High concentrations of the calmodulin antagonist trifluoperazine, inhibited both the Ca2+ uptake by the endoplasmic reticulum and by the mitochondria, while calmidazolium released Ca2+ from both compartments. In addition, trifluoperazine and calmidazolium inhibited respiration and collapsed the mitochondrial membrane potential of T. cruzi, thus indicating non-specific effects unrelated to calmodulin.125361369