To attain balanced and efficient metabolism, the genes encoding enzymes involved in intermediary metabolism must be coordinately regulated. Gross misregulation of these genes can result in deleterious metabolic imbalances, as seen in transgenic laboratory organisms with altered gene dosage. By quantifying the expression of enzymes in nine related species of Drosophila, we hoped to see patterns of coordinated changes that could be related to evolutionary constraints on regulation. The storage pools of triacylglycerols and glycogen were measured, and the activities of 12 enzymes in intermediary metabolism were also quantified. When these phenotypes were placed on the phylogeny of the Drosophila species, quantitative cladistic hypotheses could be tested. The method of independent phylogenetic contrasts provides a formal statistical test of the null hypothesis that phenotypic changes of pairs of characters on a phylogenetic tree are independent. For example, G6PD and 6PGD exhibited a wide range of activity variation among species, and interspecific comparisons suggested significant coregulation. Significant coordinated changes among clades of the Drosophila phylogeny were also seen for 6PGD-malic enzyme, glycogen synthase-fatty acid synthase, G6PD-GPDH, and 6PGD-glycogen synthase, which in most cases exhibited positive intraspecific genetic correlation. This was to be contrasted with the relation between fatty-acid synthesis and fat storage, which exhibits a strong positive correlation within D. melanogaster but no significant interspecific correlation. Limitations of inferring evolutionary constraints by contrasting intraspecific phenotypic variation to interspecific divergence are briefly discussed.