The F-box protein p45 SKP2 is the substrate-targeting subunit of the ubiquitin–protein ligase SCF SKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45 SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45 SKP2 in quiescent fibroblasts promotes p27 Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27 Kip1 mutant suppresses p45 SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45 SKP2 is important in the progression from quiescence to S phase and that the ability of p45 SKP2 to promote p27 Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45 SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27 Kip1 function.