Heart mitochondria from rats at 3 months and 23 months of age were investigated for their capacity to generate oxygen radicals and hydrogen peroxide. 2. Highest values for O^...₂-formation were obtained with submitochondrial particles freed from superoxide dismutase after the addition of succinate and antimycin A to start the reaction. Under these conditions superoxide-radical formation with mitochondria from old rats (2.54 nmol x min^-1 x mg^-1) exceeded formation rates in the young controls (1.9 nmol x min^-1 x mg^-1) by 25%. 3. A constant fraction of 20% of the radicals produced escaped quenching by intramitochondrial superoxide dismutase. This fraction was independent of the rate of radical formation: its magnitude was deduced from generation rates of hydrogen peroxide in the presence and absence of exogenous superoxide dismutase. 4. Free oxygen radicals could be obtained with intact rat-heart mitochondria as well following the addition of ATP. a system which is closer to physiological states. Formation rates of oxygen radicals observed under these conditions were similar to those seen in the particulate fractions which escaped quenching by mitochondrial superoxide dismutase. 5. Peroxidative degradation of membrane lipids was found to parallel steady-state concentrations of free oxygen radicals. 6. The results are discussed in terms of a radical-generating mechanism which functions in vive at the level of the mitochondrial electron-transferring system.