The β-cells of pancreatic islets maintain the blood D-glucose concentration within a narrow range by secreting insulin in response to rising D-glucose concentrations in the fluid bathing them. D-glucose must be transported into the β-cell and metabolized there in order to induce insulin secretion. Signals derived from the metabolism of D-glucose within β-cells increase the membrane permeability to Ca2+ and induce a rise in the cytosolic [Ca2+], which is a critical signal in the induction of insulin secretion. Upon stimulation with secretagogues including D-glucose, phospholipid hydrolytic events occur in β-cells and result in the accumulation of a variety of phospholipid-derived mediators including nonesterified arachidonic acid and arachidonate metabolites. A major fraction of the D-glucose-induced hydrolysis of arachidonate from β-cell membrane phospholipids is independent of Ca2+ influx and occurs in Ca2+-free medium, but D-glucose-induced eicosanoid release from islets does not occur when glucose metabolism is prevented by compounds which inhibit the phosphorylation of D-glucose and its entry into glycolysis. Sufficient nonesterified arachidonate accumulates in D-glucose-stimulated islets to achieve an increment in the β-cell concentration of at least 30-70 μM, and such concentrations of arachidonate induce a rise in the β-cell cytosolic [Ca2+] and amplify depolarization-induced insulin secretion. The D-glucose-induced hydrolysis of arachidonate from β-cell membrane phospholipids is mediated by a phospholipase A2 enzyme whose activity is independent of Ca2+ and stimulated by ATP and which prefers plasmalogen substrates with sn-2 arachidonoyl residues. Selective inhibition of this islet ATP-stimulated, Ca2+-independent (ASCI)-phospholipase A2 (PLA2) with a haloenol lactone suicide substrate which is sterically similar to plasmalogens suppresses the D-glucose-induced hydrolysis of arachidonate from β-cell phospholipids, the rise in β-cell cytosolic [Ca2+], and insulin secretion. Islets contain substantial amounts of plasmenylethanolamine molecular species bearing arachidonoyl residues in the sn-2 position, and these molecules are hydrolyzed more rapidly than diacyl-phospholipid substrates by islet ASCI-PLA2 in vitro and also undergo hydrolysis in intact, secretagogue-stimulated islets. Islet ASCI-PLA2 appears to constitute an important component of the β-cell D-glucose sensor apparatus.