In a previous report (Macedo, D. V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.265321995519960