Our laboratory previously demonstrated Ca2+-independent phospholipase A2gamma (iPLA2gamma) is localized to mitochondria and that iPLA2 inhibition blocks cisplatin-induced caspase-mediated apoptosis. Whereas the mitochondrial permeability transition (MPT) is a key control point for apoptosis, the role of mitochondrial iPLA2gamma in MPT has not been established. In the present study, we addressed this issue. Ca2+-induced renal cortex mitochondrial (RCM) swelling was blocked by the MPT inhibitor cyclosporine A. The R-isomer of bromoenol lactone (R-BEL), which enantiospecifically inhibits iPLA2gamma, inhibited Ca2+-induced RCM MPT, whereas S-BEL (negative control) had no effect. Ca2+ treatment resulted in a significant increase in free arachidonic acid (AA) (>50 microM) in the RCM suspension that was blocked by pretreatment with BEL. No increases in free myristic, palmitic, stearic, oleic, linoleic, or docosahexaenoic acid were detected after Ca2+ treatment. The addition of AA (18 microM) to Ca2+-treated RCM with inhibited iPLA2gamma activity restored MPT. We also determined that RCM iPLA2gamma displays higher activity against plasmenylcholine with AA in the sn-2 position than oleic acid. Ca2+ exposure significantly increased RCM iPLA2gamma activity; however, the Ca2+-induced activation of iPLA2gamma was not the result of mitochondrial membrane potential dissipation, opening of the MPT pore, or mitochondrial swelling. Taken together these findings provide strong evidence that Ca2+-induced RCM MPT is mediated by iPLA2gamma-catalyzed AA liberation.