We employed quantitative trait locus (QTL) mapping to dissect the genetic architecture of a hierarchy of functionally related physiological traits, including metabolic enzyme activity, metabolite storage, metabolic rate, and free-flight performance in recombinant inbred lines of Drosophila melanogaster. We identified QTL underlying variation in glycogen synthase, hexokinase, phosphoglucomutase, and trehalase activity. In each case variation mapped away from the enzyme-encoding loci, indicating that trans-acting regions of the genome are important sources of variation within the metabolic network. Individual QTL associated with variation in metabolic rate and flight performance explained between 9 and 35% of the phenotypic variance. Bayesian QTL analysis identified epistatic effects underlying variation in flight velocity, metabolic rate, glycogen content, and several metabolic enzyme activities. A region on the third chromosome was associated with expression of the glucose-6-phosphate branchpoint enzymes and with metabolic rate and flight performance. These genomic regions are of special interest as they may coordinately regulate components of energy metabolism with effects on whole-organism physiological performance. The complex biochemical network is encoded by an equally complex network of interacting genetic elements with potentially pleiotropic effects. This has important consequences for the evolution of performance traits that depend upon these metabolic networks.