The mechanism through which internal Ca2+ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca2+-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca2+ accumulated by the mitochondria and on the time that Ca2+ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca2+, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca2+-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca2+.