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 400muM, while Na(+)-independent Ca(2+) release was very slow at [Ca(2+)](M) below 200muM, 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 5-10mM [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 7muM, 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 7muM, but its effect was hardly appreciable at 10muM [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.