The mitochondrial inner membrane can undergo a permeability increase known as "permeability transition" elicited by Ca2+ and several other inducing agents. In general, the condition of oxidative stress acts as an inducer, at variance with antioxidants and reducing agents that inhibit the permeability transition. The action of mitochondrial respiratory substrates in preventing the permeability transition induced by Ca2+ and phosphate was examined; pyruvate, isocitrate, and glutamate proved to be particularly effective. The effect of substrates was evident also in the presence of an uncoupler, and, in addition, they were able to counteract the swelling stimulated by acetoacetate and tert-butylhydroperoxide. In the presence of various pyridine nucleotide-dependent substrates, mitochondria are able to reduce the disulfide 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to an extent far larger than that calculated from the theoretical amount of total mitochondrial thiol groups, indicating the occurrence of a catalytic system. Similarly, the enzymes of the mitochondrial matrix in the presence of either NADH or NADPH are able to reduce DTNB. The results are discussed considering the existence of a close redox communication between pyridine nucleotides and membrane thiol groups, possibly mediated by dithiols such as thioredoxin and lipoic acid.