Cells deprived of serum mitogens will either undergo immediate cell cycle arrest or complete mitosis and arrest in the next cell cycle. The transition from mitogen dependence to mitogen independence occurs in the mid- to late G(1) phase of the cell cycle and is called the restriction point. Murine Balb/c-3T3 fibroblasts deprived of serum mitogens accumulated the cyclin-dependent kinase (CDK) inhibitor p27(Kip1). This was correlated with inactivation of essential G(1) cyclin-CDK complexes and with cell cycle arrest in G(1). The ability of specific mitogens to allow transit through the restriction point paralleled their ability to down-regulate p27, and antisense inhibition of p27 expression prevented cell cycle arrest in response to mitogen depletion. Therefore, p27 is an essential component of the pathway that connects mitogenic signals to the cell cycle at the restriction point.