Renal ischemia was induced in the rat by clamping the renal artery for 45 minutes, and the ability of the K-ATP channel opener diazoxide (DZ) to ameliorate the renal ischemia reperfusion injury (IRI) was evaluated. In this model BUN and Cr were elevated at day two but returned closer to normal at day seven. Histological staining for reactive oxygen species (ROS) and for the presence of oxidized DNA, detected by the presence of the stable adduct, 8-hydroxy-2-deoxyguanosine (8-OHdG), was clearly positive in the cytoplasm of tubular cells, after just one hour of reperfusion and declined by day 7 after reperfusion. This finding was confirmed by ELISA methods which detected 8-OHdG in the mitochondrial fraction of kidney homogenates. Despite evidence of improved function measured by BUN and Cr on day 7, the early changes in tubules were remained alarming as we found mitochondrial DNA (mtDNA) had the common deletion, and the number of TUNEL positive tubular cells increased. There was continued activation of caspase-3, and abnormal levels of ROS were found in the mitochondrial fraction of cellular homogenates. The striking finding was that DZ provided prior to ischemia reduced or prevented both the acute and subacute deleterious effects associated with renal IRI. We conclude that excess production of ROS by mitochondria on reperfusion is a major upstream event in renal reperfusion injury; and that DZ functioned by preventing ROS accumulation in the mitochondria after IRI thereby reducing oxidative stress as measured by the presence of oxidized mtDNA and features of apoptosis.