Compounds which induce calcium efflux from calcium-loaded mitochondria generally provoke membrane leakiness. The involvement of the ADP ATP carrier in modification of mitochondrial membrane properties was studied. The addition of impermeant inhibitors of the ADP ATP carrier, namely carboxyatractylate, palmitoyl coenzyme A (in the absence of carnitine), and pyridoxal 5-phosphate, to calcium-loaded mitochondria triggered the release of accumulated calcium, the leakage of endogenous ADP, and the swelling of mitochondria. Permeant ligands, such as bongkrekic acid or ADP, showed no damaging effect on membrane permeability; in fact, they impeded the membrane perturbation which was induced by the three impermeant effectors. In addition, both bongkrekic acid and ADP were able to cancel the calcium loss and swelling resulting from the oxidation of intramitochondrial pyridine nucleotides by acetoacetate. In acetoacetate-treated mitochondria, the ADP ATP carrier was shown to be mainly in a c-state conformation (ie, the nucleotide binding site had an external orientation). It was concluded that induction of membrane leakiness by calcium ions depends on the conformational state of the adenine nucleotide carrier. The ability of intramitochondrial calcium ions to modify membrane properties is determined by the orientation of the nucleotide binding site. Only the c-state conformation allows membrane destabilization. Consequently, all compounds which stabilize the ADP ATP carrier in the c-state conformation will have a deleterious effect on calcium-loaded mitochondria.