Flying birds couple a high daily energy turnover with double-digit millimolar blood glucose concentrations and insulin resistance. Unlike mammalian muscle, flight muscle predominantly relies on lipid oxidation during locomotion at high fractions of aerobic capacity, and birds outlive mammals of similar body mass by a factor of three or more. Despite these intriguing functional differences, few data are available comparing fuel oxidation and free radical production in avian and mammalian skeletal muscle mitochondria. Thus we isolated mitochondria from English sparrow pectoralis and rat mixed hindlimb muscles. Maximal O(2) consumption and net H(2)O(2) release were measured in the presence of several oxidative substrate combinations. Additionally, NAD- and FAD-linked electron transport chain (ETC) capacity was examined in sonicated mitochondria. Sparrow mitochondria oxidized palmitoyl-l-carnitine 1.9-fold faster than rat mitochondria and could not oxidize glycerol-3-phosphate, while both species oxidized pyruvate, glutamate and malate-aspartate shuttle substrates at similar rates. Net H(2)O(2) release was not significantly different between species and was highest when glycolytic substrates were oxidized. Sonicated sparrow mitochondria oxidized NADH and succinate over 1.8 times faster than rat mitochondria. The high ETC catalytic potential relative to matrix substrate dehydrogenases in sparrow mitochondria suggests a lower matrix redox potential is necessary to drive a given O(2) consumption rate. This may contribute to preferential reliance on lipid oxidation, which may result in lower in vivo reactive oxygen species production in birds compared with mammals.