Dietary sucrose and ethanol are potent modulators of sn-glycerol-3-phosphate dehydrogenase (GPDH) in the third instar larvae of Drosophila melanogaster. When added to modified Sang's medium C, 428 mM ethanol and 146 mM sucrose each increased the GPDH tissue activity more than 90% and GPDH cross-reacting material (CRM) more than 50% over the levels found in larvae fed the 14.6 mM sucrose control diet. When fed together, ethanol and sucrose exerted synergetic effects on GPDH activity and CRM. The activity of glycerol-3-phosphate oxidase was also stimulated by dietary ethanol and sucrose, indicating that the glycerol-3-phosphate cycle was operating in the larvae. Dietary ethanol caused similar shifts in the NADH:NAD+ ratio in wild-type and Gpdh null larvae, suggesting that the maintenance of the cofactor equilibrium is not the primary function of GPDH in larvae. Increases in triacylglycerol content associated with the administration of ethanol and sucrose to larvae suggested that the formation of glycerol-3-phosphate for use in lipid synthesis is an important function of GPDH in larvae. Because ethanol is a constituent of the natural diet of D. melanogaster, nutritional modulation of GPDH is postulated to be an important aspect of the adaptation of the species to its environment.