The objective of this study was to elucidate the possible nature of the mechanism underlying the widely observed phenomenon that the rate of H2O2 production by mitochondria increases during the aging process, using flight muscle mitochondria of the male housefly as a model system. The protein carbonyl content of mitochondria increased linearly with age of the flies, and was also inversely associated with the life expectancy of flies. Exposure of flies to 100% oxygen caused a progressive increase in the level of mitochondrial carbonyl content. The rate of H2O2 release by such oxidatively damaged mitochondria was higher than the controls. Similarly, X-irradiation of submitochondrial particles simultaneously resulted in increased rate of H2O2 production and elevated level of carbonyl content. Results of this and previous studies indicate that oxidative damage to mitochondrial membranes may be responsible for the age-related increase in mitochondrial H2O2 generation.