The p53 tumor suppressor is regulated by the MDM2 oncoprotein. Overexpression of MDM2 maintains p53 at low levels and contributes to the functional inactivation of p53 in a subset of tumors. We found that treatment with roscovitine and olomoucin, which were originally developed as cyclin-dependent kinase (CDK) inhibitors, can efficiently stabilize and activate nuclear p53 in tumor cells with MDM2 amplification or cytoplasmic p53. These inhibitors block the degradation of p53 without affecting p53-MDM2 binding and the nuclear shuttling function of p53 and MDM2. Roscovitine also induces stabilization of the p53 Ala-315 mutant, indicating that it does not act by regulating the CDK phosphorylation of serine 315. Roscovitine induces down-regulation of MDM2 expression at both protein and mRNA levels. Ectopic expression of MDM2 can abrogate the ability of roscovitine to induce p53 stabilization. Low concentrations of roscovitine cooperate with the DNA-damaging agent camptothecin to activate p53 in a synergistic fashion. These results show that the small molecule CDK inhibitors can be used to activate p53 through their potent inhibitory effect on MDM2 expression and may be useful as sensitizing agents for other DNA-damaging drugs.