Dysfunction of mitochondria after oxidation of endogenous NAD(P)H, especially after calcium accumulation, has been abundantly reported, but the causes of membrane perturbations did not receive a full explanation. In light of several additional observations reported in this study, we propose a general scheme which shows the sequential processes that are likely involved in the appearance of calcium-induced membrane leakiness. Addition of acetoacetate, oxaloacetate, or ketomalonate to rotenone-treated mitochondria led to a massive oxidation of both NADH and NADPH. Under these conditions, stimulation of fatty acid oxidation could be observed. This process was shown to be accompanied by a reduction of intramitochondrial NADP+. The reduction of NADP+ was inhibited by uncouplers, electron transfer inhibitors and N,N'-dicyclohexylcarbodiimide. It was thus probably catalyzed by the mitochondrial transhydrogenase. Oxidation of pyridine nucleotides in the presence of acetoacetate induced (i) a slight decrease in the number of sulfhydryl groups reactive with N-ethylmaleimide (but no change in the amount of intramitochondrial reduced glutathione) and (ii) modifications of the kinetics and the orientation of the ADP/ATP carrier. In the presence of calcium ions, acetoacetate-stimulated fatty acid oxidation promoted an extensive swelling of mitochondria. Uptake of calcium ions into the matrix was a critical factor for triggering the swelling. Thiols, if they were added at a sufficiently high concentration, suppressed the swelling. Also ligands of the ADP/ATP carrier which stabilized the m-state conformation of the protein, exerted an efficient protective action. Three essential interacting factors emerge from this study: (i) The crucial role of the ADP/ATP carrier orientation in promoting the calcium-induced membrane destabilization. More precisely, it has been shown that the ADP/ATP carrier adopts the c-state conformation (i.e., nucleotide binding site facing the cytoplasm) during fatty acid oxidation. (ii) The modification of a very small number of sulfhydryl groups of mitochondrial protein. These groups are probably in an oxidized state when the level of reduced pyridine nucleotides is low. (iii) The prevailing role of the transhydrogenase, the function of which is also intimately associated with fatty acid oxidation. After energization, transhydrogenase can hinder thiol oxidation and therefore partially protect the membrane structure.