d-Glucose induces insulin secretion from β-cells of pancreatic islets by processes involving glycolytic metabolism and generation of ATP. Glucose also induces hydrolysis of β-cell membrane phospholipids and accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in β-cell Ca2+ concentration that is a critical signal in the induction of insulin secretion. Glucose-induced hydrolysis of arachidonate from β-cell phospholipids is mediated in part by an ATP-stimulated, Ca2+-independent (ASCI)-phospholipase A2 (PLA2), which, in vitro, prefers plasmalogen over diacylphospholipid substrates, but it is not known whether islets contain plasmalogens. We have identified and quantitated the major species of arachidonate-containing phospholipids in pancreatic islets by high-performance liquid chromatographic and mass spectromètric analyses. Arachidonate has been found to constitute 30% of the total islet glycerolipid fatty acyl mass. Ethanolamine phospholipids contain 30% of total islet arachidonate, and 44% of that amount resides in three plasmenylethanolamine molecular species with residues of palmitic, oleic, or stearic aldehydes in the sn-1 position. These endogenous islet plasmenylethanolamine species are hydrolyzed more rapidly than phosphatidylethanolamine species by islet ASCI-PLA2 in vitro and are also hydrolyzed in intact islets stimulated with secretagogues. ASCI-PLA2-catalyzed hydrolysis of islet plasmenylethanolamine species in vitro is inhibited by a selective haloenol lactone suicide substrate (HELSS) which is sterically similar to plasmalogens, and HELSS also inhibits all temporal phases of both eicosanoid release and insulin secretion from secretagogue-stimülated pancreatic islets. Islet β-cell ASCI-PLA2-catalyzed hydrolysis of arachdionate from endogenous plasmenylethanolamine substrates may be an intermediary biochemical event in the induction of insulin secretion.