Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca²⁺andt-butyl hydroperoxide or inorganic phosphate, but not 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or phenylarsine oxide, are inhibited by the local anesthetic dibucaine. Dibucaine promotes an inhibition of the Ca²⁺-induced increase in mitochondrial H₂O₂generation measured by the oxidation of scopoletin in the presence of horseradish peroxidase. This decrease in mitochondrial H₂O₂generation may be attributed to the reduction of Ca²⁺binding to the membrane induced by dibucaine, as assessed by measuring⁴⁵Ca²⁺binding to the mitochondrial membrane. Mg²⁺also inhibited Ca²⁺binding to the mitochondrial membrane, mitochondrial swelling, membrane protein thiol oxidation, and H₂O₂generation induced by Ca²⁺. Together, these results demonstrate that the mechanism by which dibucaine and Mg²⁺inhibit mitochondrial permeability transition is related to the decrease in reactive oxygen species generation induced by Ca²⁺-promoted alterations of inner mitochondrial membrane properties.