Nitric oxide (NO) is an important bioactive molecule involved in a variety of physiological and pathological processes. At the same time, NO is also an inducer of stress signaling, owing to its ability to damage proteins and DNA. NO was reported to be a potent activator of the p53 tumor suppressor protein. However, the mechanisms underlying p53 activation by NO remain to be elucidated. We report here that NO induces the accumulation of transcriptionally active p53 in a variety of cell types and that NO signaling to p53 does not require ataxia telangiectasia-mutated (ATM), poly(ADP-ribose) polymerase 1, or the ARF tumor suppressor protein. In mouse embryonic fibroblasts, NO elicits a down-regulation of Mdm2 protein levels that precedes the rise in p53. NO-induced down-regulation of Mdm2 protein but not its mRNA also occurs in several p53-deficient cell types and is thus p53-independent. The drop in endogenous Mdm2 levels following NO treatment is accompanied by a corresponding reduction in the rate of p53 ubiquitination. Thus, the down-regulation of Mdm2 by NO is likely to contribute to the activation of p53.