p21CIP1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. The study explores the interaction of p21 with various Cdk complexes. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5–15 nM) but is less effective against Cdc2/cyclin B (Ki ~400 nM) and Cdk5/p35 (Ki >2 μM), and does not associate with Cdk7/cyclin H. Overexpression of p21 arrests cells in G1, indicating its selective inhibition of G1/S Cdk/cyclin complexes. Association of p21 with Cdks is enhanced by cyclin binding, a property shared by the structurally related inhibitor p27. In normal diploid fibroblasts, the majority of active Cdk2 is associated with p21, which can be fully inhibited by exogenous p21. These findings suggest that p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.p21CIP1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. The study explores the interaction of p21 with various Cdk complexes. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5–15 nM) but is less effective against Cdc2/cyclin B (Ki ~400 nM) and Cdk5/p35 (Ki >2 μM), and does not associate with Cdk7/cyclin H. Overexpression of p21 arrests cells in G1, indicating its selective inhibition of G1/S Cdk/cyclin complexes. Association of p21 with Cdks is enhanced by cyclin binding, a property shared by the structurally related inhibitor p27. In normal diploid fibroblasts, the majority of active Cdk2 is associated with p21, which can be fully inhibited by exogenous p21. These findings suggest that p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.