A brief, 3 min period of global forebrain ischemia in the rat, induced by bilateral common carotid occlusion combined with hypotension, confers resistance to hippocampal pyramidal neurons against a subsequent 10 min ischemia, which is normally lethal to these cells. The molecular mechanisms underlying this ischemic preconditioning, or tolerance, are poorly understood. The tumor suppressor p53 is a transcription factor implicated in neuronal death following various insults, including cerebral ischemia. p53 is activated in response to cellular stress, e.g. hypoxia and DNA damage. Using in situ hybridization, we investigated the hippocampal mRNA expression of p53, and two of its target genes, p21(WAF1/Cip1) and the recently cloned PAG608/Wig-1, in a two-vessel occlusion model of ischemic preconditioning. We also evaluated changes in the protein levels of p53 and PAG608/Wig-1 using immunohistochemistry. The mRNA levels of all three genes increased in the ischemia sensitive CA1 region both following 3 min (non-lethal) preconditioning and 10 min of (lethal) nonconditioned ischemia. In contrast, after 10 min of ischemia preconditioned by a 3 min ischemic insult 48 h earlier, no upregulation of these genes was detected in the CA1. Following 10 min of nonconditioned ischemia, increased neuronal immunostaining of p53 and PAG608/Wig-1 was observed in the hippocampus, which was less pronounced following 3 min of preconditioning ischemia and 10 min of preconditioned ischemia. Our results demonstrate that activation of p53 and its response genes p21(WAF1/Cip1) and PAG608/Wig-1 occurs in the brain following lethal as well as non-lethal ischemic insults, and that ischemic preconditioning markedly diminishes this activation. Copyright 1999 Elsevier Science B.V.