Cot/Tpl-2 kinase, homologous to members of mitogen-activated protein kinase kinase kinase, was initially discovered by its capacity to promote cell transformation. Cot/Tpl-2 mRNA levels are increased during G0 to G1 phase progression in T lymphocytes, suggesting a role for this kinase later on in the cell cycle. The IL-2-dependent CTLL-2 cells were used to investigate the role of Cot kinase in G1 to S phase transition. Transient expression of Cot kinase in CTLL-2 cells increases DNA synthesis triggered by IL-2 and the transient expression of a dominant negative form of Cot kinase in CTLL-2 markedly reduces the DNA synthesis triggered by this cytokine. Cell cycle analysis of synchronized CTLL-2 stabling overexpressing Cot kinase indicates that this kinase contributes to the passage to S and G2-M phases of the cell cycle. Cot kinase reduces the levels of the cyclin kinase inhibitor p27kip, whereas bcl-xL expression is unaffected. Cot kinase also increases E2F transcriptional activity in a phosphatidylinositol 3 kinase-independent way and acts in synergy with this kinase. These data give evidence, for the first time, of the regulation of different G1 progression events by Cot kinase.