The involvement of mitochondrial Na + -Ca 2 + exchange in Ca 2 + responses to ATP was examined in rat pheochromocytoma (PC) 12 cells. Intracellular Ca 2 + ([Ca 2 + ] i ) and Na + concentrations ([Na + ] i ) were measured using fura-2 and SBFI, respectively. ATP caused concentration-dependent increases in [Ca 2 + ] i and [Na + ] i . High concentrations of ATP elicited a Ca 2 + transient followed by a slow recovery of [Ca 2 + ] i (a sustained phase) in 77% of PC12 cells. The sustained phase of Ca 2 + response appeared only when the peak Ca 2 + transient exceeded 500 nM. FCCP, a protonophore, greatly enhanced Ca 2 + responses to ATP only in cells with the sustained phase but not without this phase. The sustained phase was decreased by clonazepam and CGP37157, mitochondrial Na + -Ca 2 + exchange inhibitors, and extracellular Na + removal but not by cyclosporin A, an inhibitor of permeability transition pores. The reintroduction of Na + 3.5 min after ATP stimulation in the absence of Na + caused Na + concentration-dependent increases in [Ca 2 + ] i and [Na + ] i . The increase in [Na + ] i was correlated with that in [Ca 2 + ] i . FCCP caused a great increase in [Ca 2 + ] i 4.5 min after ATP stimulation in the absence of extracellular Na + but not in its presence, indicating that mitochondria retain Ca 2 + in the absence of Na + . These results suggest that ATP causes a large increase in [Ca 2 + ] i which was sequestered in mitochondria and that the sustained phase of Ca 2 + response to ATP are mainly due to the release of mitochondrial Ca 2 + through Na + -Ca 2 + exchangers in PC12 cells.