The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura-2 imaging. Following KCl depolarization, addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP-releasable pool was found in nondepolarized cells. Although the KCl-evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCl depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine-123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP-releasable pool was found subsequent to KCl depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCl-evoked Ca2+ elevation is decreased. It is concluded that the CCCP-induced increase in cytoplasmic Ca2+ response to KCl is due to inhibition of nonmitochondrial ATP-dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage-activated Ca2+ channel activity.