Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.