Inner membrane mitochondria undergo a permeability increase elicited after the opening of a nonspecific pore due to supraphysiological matrix Ca2+ load, and the presence of an inducer. Multiple inducers have been used to promote the transition in permeability; among them are carboxyatractyloside (CAT) and reactive oxygen-derived species. In contrast, inhibitors such as ADP and cyclosporin A have been commonly used. In this work, we show that the opening or closure of the nonspecific pore depends on the cationic composition of the incubation medium. It was found that when mitochondria were incubated in either 125 mM KCl or 125 mM LiCl, ADP was essential to maintain selective membrane permeability. Interestingly, the nucleotide was not required when the medium contained 125 mM NaCl. Furthermore, it was established that CAT promotes membrane leakage in K(+)- or Li(+)-incubated mitochondria, while it failed to do so in Na(+)-incubated mitochondria. Evidence is also presented on the ability of Na+ to induce resistance in mitochondria against membrane damage by oxidative stress. Mitochondrial Ca2+ discharge, swelling, and transmembrane electric gradient were analyzed to establish permeability transition. It is concluded that the protection provided by Na+ was accomplished by inducing matrix K+ depletion, which, in turn, diminished the free fraction of matrix Ca2+.