Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.