The nuclear enzyme poly (ADP-Ribose) Polymerase (PARP) has been implicated in ischemia-reperfusion injury in many tissues under normothermic conditions. The aim of this study was to determine if PARP contributes to mechanisms of hypothermic ischemia-reperfusion injury that occurs when kidneys are cold stored for transplantation. Cortical tissue slice PARP enzyme activity rose significantly with prolonged cold storage and was dependant on both reperfusion and preservation quality. However, prior exposure to warm ischemia abrogated this increase. PARP protein increased with cold storage but was not dependent on reperfusion. PARP enzyme activity rose quickly after reperfusion in buffer and was not different when whole blood was used. Addition of exogenous hydrogen peroxide (3 mM) to normal renal slices significantly increased PARP activity over 4 hours in cortex but not in the medulla, but the medulla basal PARP synthesis rate was 5 times higher than the cortex. However, the reactive oxygen species (ROS) inhibitors catalase (2,000 U/ml), Trolox (200 µM), and DMSO (15 mM) did not reduce reperfusion-induced PARP activity in cold stored cortical slices. Finally, PARP inhibitors potentiated preservation injury in isolated canine proximal renal tubules. In conclusion, canine renal PARP enzyme activity rises with prolonged cold storage after reperfusion and may play a protective rather than an injurious role in hypothermic preservation for transplantation. ROS are sufficient but not necessary to activate PARP under these conditions.