The objective of this study was to elucidate the anti-oxidative roles of coenzyme Q (CoQ) and alpha-tocopherol in mitochondrial membranes by determining whether CoQ directly scavenges peroxyl- and alkoxyl-radicals or indirectly regenerates alpha-tocopherol during autooxidation of mitochondrial membranes. A comparison of the interaction between alpha-tocopherol and CoQ during autooxidation was made between bovine and rat heart mitochondria, which differ approximately 15-fold in their alpha-tocopherol content. Autooxidation of both bovine and rat heart mitochondria resulted in the formation of thiobarbituric-acid-reactive substances and protein carbonyls; however, the differences in the autooxidizability of mitochondria between rat and bovine heart mitochondrial membranes were relatively minor. Supplementation of rat heart mitochondria with succinate caused reduction of CoQ to ubiquinol while alpha-tocopherol concentration remained unaffected during autooxidation. In contrast, in the absence of succinate, CoQ was present in the oxidized form (ubiquinone) and the mitochondrial membranes were depleted of alpha-tocopherol. CoQ concentrations remained unchanged over time irrespective of the presence or absence of succinate. In the absence of succinate, autooxidation of bovine SMPs, supplemented with different amounts of alpha-tocopherol, was inversely related to the amount of alpha-tocopherol, whereas in the presence of succinate autooxidation was greatly reduced. Results of this study indicate that during autooxidation of mitochondria, alpha-tocopherol acts as the direct radical scavenger, whereas ubiquinol regenerates alpha-tocopherol.