Pyruvate cycling was examined in the insect Manduca sexta L. (2-C-13)pyruvate was injected into 5th instar larvae maintained on a semisynthetic high sucrose, low sucrose, or sucrose-free diet. Pyruvate cycling and gluconeogenesis were determined horn the distribution of C-13 in blood metabolites, including trehalose, the blood sugar of insects, and alanine, Pyruvate cycling was evident from the C-13 enrichment of alanine C3, synthesized by transamination of pyruvate following carboxylation to oxaloacetate and cycling through phosphoenolpyruvate, Based on the relative C-13 enrichments of alanine C2 and C3, insects maintained on the high sucrose diet displayed higher levels of cycling than. insects on the other diets. Insects on all the diets, when subsequently starved, displayed low levels of cycling. Gluconeogenesis was evident in insects on sucrose-free or low sucrose diets from the selective C-13 enrichment in trehalose, The level of gluconeogenesis relative to glycolysis was indicated by the C-13 enrichment of trehalose C6 and alanine C3, both enrichments metabolically derived in the same manner. Insects starved after maintenance on the sucrose-free or low sucrose diets remained glucogenic. Insects on the high sucrose diet were not glucogenic, and subsequent starvation did not induce gluconeogenesis, The results indicate that pyruvate kinase plays a critical role in regulating the gluconeogenic/glycolytic balance, and that inhibition of pyruvate kinase is a principal regulatory event during induction of de nova trehalose synthesis. Gluconeogenesis failed to maintain homeostatic levels of blood trehalose, supporting the conclusion, that blood sugar level may be important for mediating nutrient intake. Possible factors involved in the regulation of gluconeogenesis in insects are discussed.