Rat pancreatic islets contain aspartate aminotransferase and malate dehydrogenase, enzymes necessary for the malate aspartate hydrogen shuttle, in both the cytosolic and mitochondrial fractions. When supplied with glutamate and malate, intact mitochondria from islets synthesized aspartate, indicating the mitochondrial segment of the malate aspartate shuttle was reconstituted. Aspartate synthesis was inhibited by aminooxyacetate, an inhibitor of aspartate aminotransferase, and also by butylmalonate, an inhibitor of malate transport across the mitochondrial inner membrane. Each inhibitor decreased insulin release and CO₂ production from glucose by pancreatic islets in a concentration-dependent manner. It is concluded that the malate aspartate shuttle may be involved in stimulus secretion coupling for glucose-induced insulin release.