The cyclin-dependent kinase (CDK) inhibitor p21 is induced by the tumor suppressor gene product p53 and is thought to be important for the arrest of the cell cycle following DNA damage. Here we have investigated the contribution of p21 in inhibiting different cyclin-CDK complexes that drive different cell cycle transitions following UV irradiation-induced DNA damage in normal human fibroblasts and immortalized rodent fibroblasts. When cells were exposed to a low dose of UV irradiation, both p53 and p21 were induced; the protein kinase activities associated with Cdc2, Cdk2, and Cdk4 were inhibited; and there was a good correlation between their inhibition and binding to p21. p21 alone is likely to be sufficient for the inhibition of Cdk2 because all the cyclin-complexed forms of Cdk2 were associated with p21 after irradiation. In contrast, only a small proportion of Cdk4 and Cdc2 was complexed with p21, although the level of Cdk4 associated with either p21 or p27 was increased after irradiation. Furthermore, recombinant p21 added to an unirradiated cell lysate at the same level as that induced by irradiation damage inhibited only the kinase activity associated with Cdk2. Cdc2 is likely to be inhibited by Thr-14/Tyr-15 phosphorylation after irradiation because Cdc2 was tyrosine-phosphorylated, and recombinant Cdc25 was able to increase its kinase activity significantly. Taken together, these results suggest that different CDKs are inhibited by different mechanisms following UV-induced DNA damage: Cdk2 is inhibited by the elevated level of p21; Cdk4 is inhibited by cooperation of p21 with other CDK inhibitors, like p27, and possibly by phosphorylation; and Cdc2 is inhibited by Thr-14/Tyr-15 phosphorylation. It is likely that these underlying mechanisms that inactivate CDKs are similar for other kinds of DNA damage.