The inetic properties of exchangeable Ca2+ in isolated guinea-pig heart mitochondria were studied at 25°C in the presence of 0.9 mM free Mg2+, ATP, phosphate ions and 0.4 - 0.5 μM free Ca2+ using a 45Ca2+ exchange technique. The simplest system which was found to be consistent with the data was one in which two kinetically-distinct compartments of exchangeable Ca2+ are present in the mitochondria. In the presence of 6 mM Na and at 0.4 μM free Ca2+, the fractional transfer rates for the transport of Ca2+ from these compartments were found to be 0.6 and 0.05 min-1 and the quantities of exchangeable Ca2+ 0.04 and 0.2 μmol/g wet wt heart, respectively. The amount of 45Ca2+ exchanged increased when the concentration of inorganic phosphate was increased, and decreased slightly when the concentration of free Mg2+ was increased from 1 mM to 3 mM. The flux of Ca2+ across the boundaries of both compartments was inhibited by an increase in the concentration of extramitochondrial Na+. The contribution of mitochondrial Ca2+ to compartments of kinetically-distinct exchangeable Ca2+ observed in intact cardiac muscle is briefly discussed.