Cy or RXL motifs have been previously shown to be cyclin binding motifs found in a wide range of cyclin-Cdk interacting proteins. We report the first kinetic analysis of the contribution of a Cy motif on a substrate to phosphorylation by cyclin-dependent kinases. For both cyclin A-Cdk2 and cyclin E-Cdk2 enzymes, the presence of a Cy motif decreased the Km(peptide) 75-120-fold while the kcat remained unchanged. The large effect of the Cy motif on the Km(peptide) suggests that the Cy motif and (S/T)PX(K/R) together constitute a bipartite substrate recognition sequence for cyclin-dependent kinases. Systematic changes in the length of the linker between the Cy motif and the phosphoacceptor serine suggest that both sites are engaged simultaneously to the cyclin and the Cdk, respectively, and eliminate a "bind and release" mechanism to increase the local concentration of the substrate. PS100, a peptide containing a Cy motif, acts as a competitive inhibitor of cyclin-Cdk complexes with a 15-fold lower Ki for cyclin E-Cdk2 than for cyclin A-Cdk2. These results provide kinetic proof that a Cy motif located a minimal distance from the SPXK is essential for optimal phosphorylation by Cdks and suggest that small chemicals that mimic the Cy motif would be specific inhibitors of substrate recognition by cyclin-dependent kinases.