Mitochondrial metabolism is a critical component in the functioning and maintenance of cellular organs. The stoichiometry of biochemical reaction networks imposes constraints on mitochondrial function. A modeling framework, flux-balance analysis (FBA), was used to characterize the optimal flux distributions for maximal ATP production in the mitochondrion. The model predicted the expected ATP yields for glucose, lactate, and palmitate. Genetic defects that affect mitochondrial functions have been implicated in several human diseases. FBA can characterize the metabolic behavior due to genetic deletions at the metabolic level, and the effect of mutations in the tricarboxylic acid (TCA) cycle on mitochondrial ATP production was simulated. The mitochondrial ATP production is severely affected by TCA-cycle mutations. In addition, the model predicts the secretion of TCA-cycle intermediates, which is observed in clinical studies of mitochondriopathies such as those associated with fumarase deficiency. The model provides a systemic perspective to characterize the effect of stoichiometric constraints and specific metabolic fluxes on mitochondrial function.