20-hydroxyeicosatetraenoic acid (20-HETE), a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified SIN lentiviral vectors were generated to stably over express murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 hours of ATP depletion and 2 hours of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P<0.05), compared to LLC-EGFP cells. Treatment with the superoxide dismutase (SOD) mimetic, MnTMPyP (100 µM), resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor, HET-0016 (10 µM), also inhibited cytotoxicity significantly (P<0.05) in LLC-Cyp4a12 cells. DHE fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared to control cells, and that this increase was inhibited by MnTMPyP. There was a significant increase (P<0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 versus LLC-EGFP cells (P<0.05). This was abolished in the presence of HET-0016 (P<0.05) or MnTMPyP (P<0.01). These results demonstrate that 20-HETE over expression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent upon enhanced CYP4A generation of free radicals.