The newly discovered oncogenic protein gankyrin, which contains six ankyrin repeats, has been reported to be involved in the phosphorylation and degradation of the retinoblastoma gene product, Rb. Using in vitro systems, we have identified a peptide fragment of gankyrin, ¹⁷⁶LHLACDEERN¹⁸⁵, which is responsible for binding of gankyrin to Rb. We further demonstrated a different mechanism for gankyrin to facilitate the phosphorylation of Rb, by binding with cyclin-dependent kinase 4 (CDK4). This binding does not inhibit the Rb-phosphorylating kinase activity of CDK4, but it competes with p16 binding to CDK4 and counteracts the inhibitory function of p16. We then showed that binding of gankyrin to CDK4 and the consequent counter action of p16 function were not affected by the Rb-binding peptide ¹⁷⁶LHLACDEERN¹⁸⁵, indicating that the two mechanisms are independent. They also involve different structural regions of gankyrin. While the Rb-binding motif is located at the fifth ankyrin repeat, truncation mutants of gankyrin, with the first three or four ankyrin repeats remaining, are sufficient for binding to CDK4 and for counteracting the inhibitory function of p16. These results demonstrate the potential importance of gankyrin in cell cycle control and tumorigenesis and suggest an expanded INK4-CDK4/6-Rb pathway.