We studied the interaction between energy metabolism and mitochondrial biogenesis during myogenesis in C2C12 myoblasts. Metabolic rate was nearly constant throughout differentiation, although there was a shift in the relative importance of glycolytic and oxidative metabolism, accompanied by increases in pyruvate dehydrogenase activation state and total activity. These changes in mitochondrial bioenergetic parameters observed during differentiation occurred in the absence of a hypermetabolic stress. A chronic (3 day) energetic stress was imposed on differentiated myotubes using sodium azide to inhibit oxidative metabolism. When used at low concentrations, azide inhibited more than 70% of cytochrome oxidase (COX) activity without changes in bioenergetics (either lactate production or creatine phosphorylation) or mRNA for mitochondrial enzymes. Higher azide concentrations resulted in changes in bioenergetic parameters and increases in steady state COX II mRNA levels. Azide did not affect mtDNA copy number or mRNA levels for other mitochondrial transcripts, suggesting azide affects stability, rather than synthesis, of COX II mRNA. These results indicate that changes in bioenergetics can alter mitochondrial genetic regulation, but that mitochondrial biogenesis accompanying differentiation occurs in the absence of hypermetabolic challenge.