The protein p27Kip1 is an inhibitor of cell division1. An increase in p27 causes proliferating cells to exit from the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division. Abnormally low amounts of p27 are associated with pathological states of excessive cell proliferation, especially cancers. In normal and tumour cells, p27 is regulated primarily at the level of translation and protein turnover. Phosphorylation of p27 on threonine 187 (T187) by cyclin-dependent kinase 2 (Cdk2) is thought to initiate the major pathway for p27 proteolysis. To critically test the importance of this pathway in vivo, we replaced the murine p27 gene with one that encoded alanine instead of threonine at position 187 (p27T187A). Here we show that cells expressing p27T187A were unable to downregulate p27 during the S and G2 phases of the cell cycle, but that this had a surprisingly modest effect on cell proliferation both in vitro and in vivo. Our efforts to explain this unexpected result led to the discovery of a second proteolytic pathway for controlling p27, one that is activated by mitogens and degrades p27 exclusively during G1.