Abstract We have investigated the kinetics of mitochondrial Ca 2+ influx and efflux and their dependence on cytosolic [Ca 2+] and [Na +] using low-Ca 2+-affinity aequorin. The rate of Ca 2+ release from mitochondria increased linearly with mitochondrial [Ca 2+] ([Ca 2+] M). Na +-dependent Ca 2+ release was predominant al low [Ca 2+] M but saturated at [Ca 2+] M around 400 μM, while Na +-independent Ca 2+ release was very slow at [Ca 2+] M below 200 μM, and then increased at higher [Ca 2+] M, perhaps through the opening of a new pathway. Half-maximal activation of Na +-dependent Ca 2+ release occurred at 5–10 mM [Na +], within the physiological range of cytosolic [Na +]. Ca 2+ entry rates were comparable in size to Ca 2+ exit rates at cytosolic [Ca 2+] ([Ca 2+] c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca 2+] c. As a consequence, the presence of [Na +] considerably reduced the rate of [Ca 2+] M increase at [Ca 2+] c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca 2+] c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca 2+] M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca 2+ buffering, and comparison of our results with data on total mitochondrial Ca 2+ fluxes indicate that the mitochondrial Ca 2+ bound/Ca 2+ free ratio is around 10- to 100-fold for most of the observed [Ca 2+] M range and suggest that massive phosphate precipitation can only occur when [Ca 2+] M reaches the millimolar range.