A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2β) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β-cells, and other sources. Proposed iPLA2β functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β-cells. To further examine iPLA2β functions in β-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2β activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA2β cDNA. The iPLA2β-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2β -overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2β. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2β-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2β-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2β plays a signaling role in β-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.