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Although crystal structural analysis of cyclin A/cyclindependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 3 10 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3 10 helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3 10 helix regions of p21 and p27 were not required, and cyclinand Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Sitedirected mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3 10 helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3 10 helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3 10 helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G 1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2 activities. Thus the 3 10 helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.