Phosphorylation of the cyclin-dependent kinase inhibitor p27Kip1 has been thought to regulate its stability. Ser10 is the major phosphorylation site of p27Kip1, and phosphorylation of this residue affects protein stability. Phosphorylation of p27Kip1 on Ser10 has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27Kip1 protein was translocated from the nucleus to the cytoplasm at the G0-G1 transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27Kip at the G0-G1 transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27Kip1 export, whereas substitution of Ser10 with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27Kip1 on Ser10 is required for its binding to CRM1 and for its subsequent nuclear export.