Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and t-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 H2O2 generation measured by the oxidation of scopoletin in the presence of horseradish peroxidase, This decrease in mitochondrial H2O2 generation may be attributed to the reduction of Ca2+ binding to the membrane induced by dibucaine, as assessed by measuring Ca-45(2+) binding to the mitochondrial membrane. Mg2+ also inhibited Ca2+ binding to the mitochondrial membrane, mitochondrial swelling, membrane protein thiol oxidation, and H2O2 generation 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. (C) 1998 Academic Press.35917781