The ontogeny of pectoralis muscle bioenergetics was studied in growing Adélie penguin chicks during the first month after hatching and compared with adults using permeabilized fibers and isolated mitochondria. With pyruvate/malate/succinate or palmitoyl-carnitine as substrates, permeabilized fiber respiration markedly increased during chick growth (3-fold) and further rose in adults (1.4-fold). Several markers of muscle fiber oxidative activity (cytochrome oxidase, citrate synthase, hydroxyl-acyl-CoA dehydrogenase) increased 6-19 fold with age together with large rises in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial content (3-5 fold) and oxidative activities (1.5-2.4 fold). The proportion of IMF relative to SS mitochondria increased with chick age but markedly dropped in adults. Differences in oxidative activity between mitochondrial fractions were reduced in adults as compared with hatched chicks. Extrapolation of mitochondrial to muscle respirations revealed similar figures with isolated mitochondria and permeabilized fibers with carbohydrate-derived but not with lipid-derived substrates suggesting diffusion limitations of lipid substrates with permeabilized fibers. Two immunoreactive fusion proteins, Mitofusin 2 (Mfn2) and Optic Atrophy 1 (OPA1), were detected by western blots on mitochondrial extracts and their relative abundance increased with age. Muscle fiber respiration was positively related with Mfn2 and OPA1 relative abundance. Present data showed by two complementary techniques large ontogenic increases in muscle oxidative activity that may enable birds to face thermal emancipation and growth in childhood and marine life in adulthood. The concomitant rise in mitochondrial fusion protein abundance suggests a role of mitochondrial networks in the skeletal muscle processes of bioenergetics that enable penguins to overcome harsh environmental constraints.