The amount of readily exchangeable Ca2+in mitochondria of an isolated working rat heart is less than 10 ng-ions/g heart. We therefore conclude that either no Ca2+enters mitochondria or that the Ca2+which does enter is removed continuously. Using Sr2+and Mn2+we obtained evidence that the mitochondrial Na+-Ca2+exchanger was indeed operational in releasing metal from mitochon-dria of the heart. When Ca2+in the perfusate was replaced by Sr2*, we found that a significant amount of Sr2+(approximately 100 ng-ions/g heart) entered mitochondria. When the heart then was returned to a Ca2+-containing perfusate, over 80% of the Sr2+was washed out of mitochondria within 30 seconds. When low levels of Mn2+were added to the perfusate, we found that Mn accumulated in mitochondria irreversibly. This is evidence for the operation of the Na +-Ca2+exchanger because Na was found to release Ca2+and Sr2+but not Mn from isolated rat heart mitochondria. Our estimates indicate that when the Na +-Ca2+exchanger is maximally operative, as in the Sr^-perfused heart, the flux of Sr2+through mitochondria is at most 10% of the total flux needed for the activation of contraction. The low level of Ca2+in the mitochondria of Ca -perfused hearts suggests a much smaller flux of through the mitochondria in this case. We therefore conclude that mitochondria play little if any role in the beat-to-beat regulation of normal Ca2+fluxes in the rat heart.Circ Res 47: 721-727, 1980