Although Skp2 has been thought to mediate the degradation of p27 at the G₁-S transition, Skp2^−/− cells exhibit accumulation of p27 in S-G₂ phase with overreplication. We demonstrate that Skp2^−/−p27^−/− mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2^−/− mice entered the endoduplication cycle after mitogenic stimulation, whereas this phenotype was not apparent in Skp2^−/−p27^−/− mice. Cdc2-associated kinase activity was lower in Skp2^−/− cells than in wild-type cells, and a reduction in Cdc2 activity was sufficient to induce overreplication. The lack of p27 degradation in G₂ phase in Skp2^−/− cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G₂-M progression by mediating the degradation of p27.