We have previously reported that treatment of isolated rat heart mitochondria with the free radical-generating system Cu2+/tert-butylhydroperoxide produces striking changes in the adenine nucleotide translocase (ANT) of the inner membrane. These changes include a small increase in apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by its disappearance from the polypeptide profile upon further oxidant treatment (Zwizinski and Schmid (1992) Arch. Biochem. Biophys. 294, 178-183). In order to characterize its peroxidative modification in more detail, we have purified rat heart ANT and prepared polyclonal antibody against it. Using this antibody, we have observed that increasing oxidant treatment results in a gradual increase in the ANT protein's apparent molecular weight by up to 1 kDa. The progressive nature of the molecular weight shift, which parallels the generation of thiobarbituric acid reactive substances, supports the hypothesis that this phenomenon may be the result of covalent addition of increasing amounts of lipid peroxidation products. Strong oxidative treatment of cardiac mitochondria also causes fragmentation and polymerization of the ANT protein. However, Western blot analysis showed that a major portion of the original ANT survives even extensive oxidation as a distinct, modified protein. Therefore, the almost complete disappearance of ANT from Coomassie-stained gels appears to be the result of cross-linking and fragmentation reactions, as well as a decreased efficiency of the Coomassie staining. Because a measurable molecular weight shift of ANT occurs at the mildest oxidative treatment tested (resulting in the production of only 1.1 nmol malondialdehyde/mg protein), it may be relevant as a parameter of myocardial ischemia-reperfusion injury.