We have determined the minimal portion of the retinoblastoma protein (Rb) that can serve as an efficient substrate for in vitro phosphorylation by cdk4 kinase-D1 cyclin. Kinetic measurements indicate that in vitro, a 15-kDa fragment that represents the C-terminus of Rb can serve equally well as a substrate when compared with the larger 56-kDa fragment of Rb, which contains the A, B and C domains. By comparison, peptide substrates appear to be 1000-fold less efficient. Furthermore, mutational analysis indicates that not all of the five phosphorylation sites within this minimal C domain are phosphorylated equally by cdk4/D1. Ser795 is the preferred phosphorylation site, whereas the four remaining sites Ser807, Ser811, Thr821 and Thr826 are phosphorylated to a much lesser degree. Truncations of the C domain from the carboxy terminus indicate that almost all of this domain is required for efficient phosphorylation. These data suggest that the structural context of the phosphorylation site within the substrate is critical for its phosphorylation by the cdk4/D1 kinase.