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.