Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin-dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for maintaining the orderly progression of the cell cycle. The B domain of Rb contains a site that recognizes and binds the LXCXE motif found in D-type cyclins. This interaction is important for phosphorylation of Rb by cdk4/D1, although in vitro the Rb C domain alone is efficiently phosphorylated by cdk4/D1. A mutation in the C domain of Rb, L901Q, has been identified that completely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contrast, the L901Q mutation has no effect on phosphorylation by either cyclin E/cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and cdk4/D1 is specific. Introduction of the L901Q mutation into Rb containing the A, B, and C domains results in phosphorylation becoming predominantly dependent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site within the C domain. The L901 binding site can supplant the LXCXE binding site for the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811. Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the isolated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in Rb and suggests a conservation of this C domain binding motif for cyclin D1/cdk4 kinase among the Rb family of proteins.