OBJECTIVE To investigate the role of phospholipase A(2) (PLA(2)) in reperfusion injury of the kidney in an in vivo animal model, renal mitochondrial PLA(2) activity was measured under three different conditions. METHODS Male Wistar rats (n = 72) anesthetized with pentobarbital underwent renal ischemia surgically for 45 min and were reperfused for the indicated time (renal ischemia/reperfusion). Treatments included reperfusion for various predetermined periods (phase 1), exposure to hyperbaric oxygen (phase 2), and administration of reactive oxygen species (ROS) scavenger (phase 3). Thereafter, each kidney was harvested, and mitochondrial PLA(2) activity was measured by a radioisotope technique. RESULTS Ischemia/reperfusion resulted in time-related PLA(2) activation in the renal mitochondria up to 48 h of reperfusion after renal ischemia. Renal mitochondrial PLA(2) activity was further augmented by hyperbaric oxygen exposure prior to reperfusion, whereas administration of the ROS scavengers suppressed mitochondrial PLA(2) activity. CONCLUSIONS These data suggest that ROS may play an important role in the in vivo activation of PLA(2) associated with renal ischemia/reperfusion.