Studies involving pharmacologic inhibition or transient reduction of Group VIA phospholipase A2 (iPLA2β) expression have suggested that it is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels, rates of arachidonate incorporation into phospholipids, and degradation of excess phosphatidylcholine (PC). In insulin-secreting islet β-cells and some other cells, in contrast, iPLA2β signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 β-cell lines in which iPLA2β expression is stably suppressed by small interfering RNA. Two such iPLA2β knockdown (iPLA2β-KD) cell lines express less than 20% of the iPLA2β of control INS-1 cell lines. The iPLA2β-KD INS-1 cells exhibit impaired insulin secretory responses and reduced proliferation rates. Electrospray ionization mass spectrometric analyses of PC and LPC species that accumulate in INS-1 cells cultured with arachidonic acid suggest that 18:0/20:4-glycerophosphocholine (GPC) synthesis involves sn-2 remodeling to yield 16:0/20:4-GPC and then sn-1 remodeling via a 1-lyso/20:4-GPC intermediate. Electrospray ionization mass spectrometric analyses also indicate that the PC and LPC content and composition of iPLA2β-KD and control INS-1 cells are nearly identical, as are the rates of arachidonate incorporation into PC and the composition and remodeling of other phospholipid classes. These findings indicate that iPLA2β plays signaling or effector roles in β-cell secretion and proliferation but that stable suppression of its expression does not affect β-cell GPC lipid content or composition even under conditions in which LPC is being actively consumed by conversion to PC. This calls into question the generality of proposed housekeeping functions for iPLA2β in PC homeostasis and remodeling. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.