An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H2O2) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, medium-sized mammals, birds). During succinate oxidation, H2O2 production rates were generally lower in the longer-lived species; the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, white-footed mouse, naked mole-rat, Damara mole-rat, guinea pig, baboon, little brown bat, Brazilian free-tailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H2O2 production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H2O2 production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H2O2 production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms. © 2007 Medical Research Council Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007.