We discovered that two physiologically occurring metabolic intermediates, glyceraldehyde phosphate and succinate, are potent insulin secretagogues. No other glycolytic intermediate besides glyceraldehyde phosphate was insulinotropic. Succinate, when added to islets as either its monomethyl or dimethyl ester to increase its cellular permeability, was also insulinotropic. In islets, as in other cell types, these esters are apparently hydrolyzed intracellularly to succinate. Unesterified succinate and other unesterified citric acid-cycle intermediates did not stimulate insulin release. Initiation of insulin release by esters of succinate suggests that mitochondrial metabolism alone is sufficient to initiate and support insulin release. However, this is specific for succinate in that esters of fumarate, pyruvate, and citrate were not insulinotropic.