Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A(2) (iPLA(2)beta) activity in pancreatic islets and insulinoma cells suggest that iPLA(2)beta participates in insulin secretion. It has also been suggested that iPLA(2)beta is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA(2)beta-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA(2)beta-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA(2)beta-null mice is virtually identical to that of wild-type mice, and no iPLA(2)beta mRNA was observed in any tissue from iPLA(2)beta-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA(2)beta-null and wild-type mice are essentially identical under normal circumstances, but iPLA(2)beta-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the beta-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA(2)beta-null mice than in wild-type mice, but PLA(2)beta-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA(2)beta-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.