Glyceraldehyde is known to stimulate insulin release. Its influence on various parameters of islet function was investigated in order to assess the possible significance of glycolsis in the insulinotropic action of glucose. In the absence of glucose, glyceraldehyde (5-20 mM), but neither dihydroxyacetone nor glycerol stimulated insulin release in rat isolated islets. The glucose-like effect glyceraldehyde (10 mM) was characterized by a shift to the left of the curve relating insulin release to glucose concentration, without any significant increase in the maximal velocity of the secretory process. In the isolated perfused rat pancreas, glyceraldehyde provoked a biphasic secretory response. Glyceraldehyde-induced insulin release was inhibited in the absence of calcium or in the presence of epinephrine, unaffected by mannoheptulose or 3,3-tetramethyleneglutaric acid, and enhanced by theophylline and cytochalism B. Glyceraldehyde also stimulated to pro-insulin biosynthesis and 45Ca net uptake by isolated islets, the latter effect being apparently due, in part at least, to inhibition of calcium outward transport across the cell membrane. At concentrations of nearly equivalent insulinotropic potency, glucose and glyceraldehyde were metabolized at rates yielding comparable output of both lactate and 14CO2. The data indicate that glyceraldehyde mimics many effects of glucose on islet function, suggesting that the insulinotropic action of glucose may be related to its metabolism through the glycolytic pathway.