Apoptosis is increasingly recognized as a major mode of cell demise after ischemic injury to the kidney. The mediators of apoptotic cell death are many and include changes in intracellular pH, calcium, free radicals, ceramide, and adenosine triphosphate (ATP) depletion. Recently, we identified guanosine triphosphate (GTP) depletion as an independent trigger for apoptotic death after chemical anoxia in vitro. We further demonstrated that GTP salvage with guanosine inhibits tubular cell apoptosis after ischemic injury in vivo. This inhibition of apoptosis was accompanied by a significant protective effect on renal function. We also showed that p53 is the mediator of apoptosis in the setting of GTP depletion and ischemic injury. Indeed, salvage of GTP with guanosine prevented the ischemia-induced increase in p53 protein. Further, pifithrin-alpha, a potent and specific inhibitor of p53, inhibited apoptosis and protected renal function with a profile similar to that seen with guanosine. Finally, the protective effects of pifithrin-alpha involved both down-regulation of the transcriptional activation of Bax and a direct inhibition of p53 translocation to mitochondria. We propose that GTP depletion and activation of p53 are major inducers of apoptotic cell death after ischemic renal injury. In this setting, guanosine and pifithrin-alpha are potent inhibitors of apoptosis and are thus potentially useful in preventing and ameliorating functional injury to the ischemic kidney.