Oxidant-induced lipid peroxidation and cell death mediate pathologies associated with ischemia-reperfusion and inflammation. Our previous work in rabbit renal proximal tubular cells (RPTC) demonstrated that inhibition of Ca2+-independent phospholipase A2(iPLA2) potentiates oxidant-induced lipid peroxidation and necrosis, implicating iPLA2in phospholipid repair. This study was conducted to identify a RPTC mitochondrial PLA2and determine the role of PLA2in oxidant-induced mitochondrial dysfunction. iPLA2activity was detected in Percoll-purified rabbit renal cortex mitochondria (RCM) and in isolated mitochondrial inner membrane fractions from rabbit and human RCM. Immunoblot analysis and inhibitor sensitivity profiles revealed that iPLA2γ is the RCM iPLA2activity. RCM iPLA2activity was enhanced in the presence of ATP and was blocked by the PKCε V1–2 inhibitor. Oxidant-induced mitochondrial lipid peroxidation and swelling were accelerated by pretreatment with R-BEL, but not S-BEL. Furthermore, oxidant treatment of isolated RCM resulted in decreased iPLA2γ activity. These results reveal that RCM iPLA2is iPLA2γ, RCM iPLA2γ is regulated by phosphorylation by PKCε, iPLA2γ protects RCM from oxidant-induced lipid peroxidation and dysfunction, and that a strategy to preserve or enhance iPLA2γ activity may be of therapeutic benefit.