Deenergized rat liver or AS-30D hepatoma mitochondria underwent extensive swelling when incubated in the presence of Ca2+ and an oxidant of mitochondrial pyridine nucleotides. This swelling was stimulated by phosphate and inhibited by ruthenium red, Mg2+ plus ATP and dibucaine. The degree of inhibition by dibucaine was shown to vary inversely with the Ca2+ concentration. The inhibition caused by ruthenium red was completely removed by the Ca2+ ionophore ionomycin indicating that the effect of the cation is mediated by internal binding sites. Since mitochondria were totally deenergized the data definitely rule out the requirement for Ca2+ cycling across the membrane in this mechanism.154934-41