In suspensions of isolated hepatocytes, two intracellular Ca2+ pools were distinguished in the presence of the metallochrome indicator arsenazo III, first by treatment with the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and then with the Ca2+ ionophore A23187. The available evidence indicates that the two pools are of mitochondrial and extramitochondrial origin. Metabolism of t-butyl hydroperoxide by hepatocytes caused release of Ca2+ from both compartments concomitant with oxidation of cellular glutathione and NADPH, which was followed by characteristic alterations in cell surface structure. When NADPH oxidation was prevented by selective inactivation of glutathione reductase, t-butyl hydroperoxide metabolism was without effect on the mitochondrial Ca2+ pool, whereas the loss from the extramitochondrial pool was accelerated. Our results suggest that different regulatory mechanisms modulate mitochondrial (NADPH-dependent) and extramitochondrial (thiol-dependent) Ca2+ compartmentation and that disturbance of normal Ca2+ homeostasis may be critical in peroxide-induced cytotoxicity.