This study was aimed at assessing the relative contributions to H(2)O(2) detoxification by glutathione peroxidase and catalase in the mitochondrial matrix of heart. For this purpose, mitoplasts from rat heart were used in order to minimize contamination with microperoxisomes, and the kinetic rate constants of both enzymatic activities were determined along with a simulation profile. Results show that the contribution of catalase to H(2)O(2) removal in heart mitochondria is not significant, even under strong oxidative conditions, such as those achieved in ischemia-reperfusion and involving extensive glutathione depletion and high H(2)O(2) concentrations. Conversely, maintenance of the steady state levels of H(2)O(2) in the heart mitochondrial matrix seems to be the domain of glutathione peroxidase. It is suggested that the physiological role of the low amounts of catalase found in heart mitochondria is related to its peroxidatic rather than catalatic activity.