Two major intracellular Ca2+ stores, the mitochondrial and nonmitochondrial (microsomes) fractions isolated from rat cerebellum exhibited a Ca2+ concentration and ATP-dependent Ca2+ accumulation. The maximal Ca2+ accumulation in mitochondria was higher than in microsomes, but the affinity of the mitochondria for Ca2+ was lower. In this study, Ca2+ accumulation within the mitochondria was energized by ATP hydrolysis. Thus, the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and the F1F0 ATP synthase inhibitor, oligomycin, blocked Ca2+ accumulation and induced the discharge of the entrapped Ca2+ in the mitochondria, whereas the metabolic inhibitor, rotenone, affected neither the Ca2+ accumulation nor discharge. On the other hand, the uniporter inhibitor, ruthenium red, blocked the mitochondrial accumulation of Ca2+, but did not cause the discharge of preloaded Ca2+. In addition, arachidonic acid (AA), sphingosylphosphorylcholine (SPC) and sphingosine (SPH) elicited the dose-dependent release of Ca2+ from microsomal stores. Although the magnitudes of the Ca2+ release induced by AA, SPC or SPH were all dependent on the presence of extravesicular Ca2+ at concentrations ranging from 0.01 to 0.1 microM Ca2+, only the AA- and SPC-evoked Ca2+ releases were insensitive to temperature. The mitochondria were more sensitive than the microsomes to the AA induced release of accumulated Ca2+. Our results indicate the existence of multiple intracellular Ca2+ stores in nerve cells which can be released by various Ca2+ mediators.