Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A(2) (PLA(2))-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA(2) isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA(2) isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA(2) Mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA(2) (iPLA(2)) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47(phox) phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA(2) inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA(2) assay, Western blotting, and PCR confirmed that there was increased iPLA(2) activity and expression in neutrophils from people with diabetes. AA (10 mu M) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA(2) in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling. The Journal of Immunology, 2010, 184: 1507-1515.