The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G 1 /S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G 2 /M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G 2 /M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.Transitions of the eukaryotic cell cycle are orchestrated by multiple protein kinases and by the transcriptional control of cell cycle regulators (1-3). Perturbations in the cell cycle process result in abnormal cell division and proliferation, the hallmark of cancer (4). Progression through the G 1 /S and G 2 /M phases of the cell cycle is regulated by CDK2-cyclin E or A and CDK1-cyclin B kinase, respectively (3). In addition, the activity of other mitotic kinases such as Polo-like kinase 1 (PLK1) 3 must be maintained for proper mitotic progression (2,5,6). Previous studies demonstrated that the polo-box domain in PLK1 acts as a phosphopeptide-binding domain and targets PLK1 to its substrates that have been "prime" or phosphorylated by CDK1 (7). The polo-box domain-mediated PLK1 recruitment is responsible for both temporal and spatial regulation of PLK1 substrates (2, 7).The mammalian Forkhead box (Fox) proteins belong to a large family of transcription factors consisting of more than 50 proteins that share homology in the winged helix DNA-binding domain (8,9). Within this extensive family, FoxM1 is a proliferation specific transcription factor (10 -14). Expression of Foxm1 is detected only in proliferating cells and is extinguished when cells undergo terminal differentiation and exit the cell cycle (10, 12, 13). Transcription of the Foxm1 locus results in three distinct splice variants that are almost identical in sequence but differ by the addition of two small exons: Foxm1b (HFH-11B) contains no additional exons, whereas the Foxm1c (Trident, WIN, or MPP2) and Foxm1a (HFH-11A) isoforms contain one and two additional exons, respectively (11)(12)(13)15)...