A common feature across all animals, including humans, is that mitochondrial bioenergetics is linked to oxidative stress, but the nature of these relationships with survival is yet to be properly defined. In this study we included 12 Drosophila simulans isofemale lines: four of each distinct mtDNA haplogroup (siI, -II, and -III). First, we investigated sequence variation in six mtDNA and 13 nuclear encoded genes (nine nuclear-encoded subunits, and the four known isoforms, of complex IV of the electron transport chain). As expected we observed high divergence among the three distinct mitotypes and greatest mtDNA variability in siII-harboring flies. In the nuclear encoded genes, no fixed amino acid differences were observed and levels of polymorphism did not differ significantly among flies harboring distinct mtDNA types. Second, 15,456 flies were included in mortality studies. We observed that mtDNA type influenced survival (siII approximately siIII > siI), flies harboring siII mtDNA had the greatest variation in mortality rates, and in all cases males were longer lived than females. We also assayed maximal rates of hydrogen peroxide (H(2)O(2)) production from complex III of the electron transport chain in mitochondria isolated from 11-day-old flies. Contrary to our prediction, rates of H(2)O(2) production tended to increase with mean survival. This result suggests that higher rates of H(2)O(2) production in younger flies may lead to an upregulation of antioxidants, age-dependent increase in the rate of H(2)O(2) production differ, and/or flies vary in their mitochondrial uncoupling. Alternatively, the whole organism may not regularly, if ever, experience maximal H(2)O(2) production rates.