The routes by which glucose-derived pyruvate is metabolized to enter mitochondrial pathways to stimulate insulin release in the pancreatic beta cell are unknown. The 14CO2 ratios assay was used to estimate the fractions of glucose-derived pyruvate that enter the citric acid cycle by carboxylation and decarboxylation in pancreatic islets. Pyruvate 14CO2 ratios were estimated with glucose, and acetate 14CO2 ratios were estimated with succinate methyl ester. Glucose and methyl succinate, which are insulin secretagogues, gave more accurate ratios than ratios estimated with pyruvate and acetate, which do not initiate insulin release. The results indicate that relatively equal amounts of pyruvate enter the citric acid cycle by carboxylation and decarboxylation.