The ubiquitin–proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1–Cullin–F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2–/– animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2–/– cells also exhibit increased accumulation of both cyclin E and p27Kip1. The elimination of cyclin E during S and G2 phases is impaired in Skp2–/– cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27Kip1 is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.