Mitochondrial production of reactive oxygen species and oxidation of cardiolipin are key events in initiating apoptosis. We reported that group VIA Ca2+-independent phospholipase A2 (iPLA2 β) localizes in and protects β-cell mitochondria from oxidative damage during staurosporine-induced apoptosis. Here, we used iPLA2β-null (iPLA2β-/-) mice to investigate the role of iPLA 2βin the repair of mitochondrial membranes. We show that islets isolated from iPLA2β-/- mice are more sensitive to staurosporine-induced apoptosis than those from wild-type littermates and that 2 wk of daily ip administration of staurosporine to iPLA2β -/- mice impairs both the animals' glucose tolerance and glucose-stimulated insulin secretion by their pancreatic islets. Moreover, the iPLA2β inhibitor bromoenol lactone caused mitochondrial membrane peroxidation and cytochrome c release, and these effects were reversed by N-acetyl cysteine. The mitochondrial antioxidant N-t-butyl hydroxylamine blocked staurosporine-induced cytochrome c release and caspase-3 activation in iPLA2β-/- islets. Furthermore, the collapse of mitochondrial membrane potential in INS-1 insulinoma cells caused by high glucose and fatty acid levels was attenuated by overexpressing iPLA 2β. Interestingly, iPLA2β was expressed only at low levels in islet β-cells from obesity- and diabetes-prone db/db mice. These findings support the hypothesis that iPLA2β is important in repairing oxidized mitochondrial membrane components (e.g. cardiolipin) and that this prevents cytochrome c release in response to stimuli that otherwise induce apoptosis. The low iPLA2β expression level in db/db mouse β-cells may render them vulnerable to injury by reactive oxygen species.