The generation of superoxide anion radicals (O2*-) and hydrogen peroxide (H2O2) during mitochondrial respiration has been widely postulated to be causally linked to the aging process. The hypothesis that a specific enhancement of mitochondrial O2*-/H2O2 catabolism would delay age-associated physiological changes and extend the lifespan was tested by simultaneous overexpression of MnSOD (manganese superoxide dismutase) and catalase, ectopically targeted to the mitochondrial matrix of transgenic Drosophila melanogaster. The increased activities of these antioxidative enzymes resulted in a decrease of mitochondrial H2O2 release and enhancement of free methionine content. The MnSOD/mitochondrial catalase transgenic flies displayed an enhanced resistance to experimental oxidative stress, induced by dietary H2O2 administration or by exposure to 100% ambient oxygen. However, the lifespan of the flies was decreased, by up to 43%, and this effect coincided with (i) an overall decrease in physical fitness, as measured by the speed of walking, and (ii) an age-related decrease in mitochondrial state 3 (ADP-stimulated) respiration. These findings support the notion that mitochondrial O2*-/H2O2 production at physiological levels is essential for normal biological processes leading to the attainment of a normal lifespan.