The hypothesis that rate of intracellular prooxidant production is associated with the rate of aging was tested by comparing the rate of H2O2 generation by mitochondria in houseflies of similar chronological but different physiological ages. Physiological age represents the life expectancy or 'nearness to death'. Average and maximum life spans of flies were extended 2-fold by the elimination of flying activity. In addition, using senescence-related loss of flight ability as a phenotypic marker of impending death, relatively short-lived and long-lived subpopulations of flies were isolated from cohort populations. Rate of H2O2 generation was measured fluorometrically in mitochondria from thoracic flight muscles using alpha-glycerophosphate as a substrate and without employing any respiratory inhibitors as is often the case in mammalian studies. The rate of mitochondrial H2O2 release was found to be associated with life expectancy or the physiological age of flies rather than the chronological age. At the same chronological age, mitochondria from flies with a shorter life expectancy had a markedly higher rate of H2O2 generation than those with a longer life expectancy. Results of this and some previous studies in this laboratory are interpreted to suggest that the rate of prooxidant generation rather than the level of antioxidant defenses may be a key correlate of the rate of aging.