Sam68, a nuclear RNA binding protein, is the Src mitotic target and specifically tyrosine phosphorylated during mitosis. It has also been demonstrated to associate with various signal transduction molecules, thereby raising the possibility of its role in cell cycle control as a modulator of the signal transduction and activation of RNA metabolism. To elucidate the physiological function, a Sam68-deficient cell line was isolated from the chicken DT40 cell line by gene disruption. The Sam68 deficient cells exhibited markedly decreased growth, and forced expression of chicken Sam68 cDNA in the mutant cells restored the cell growth. Cell cycle analysis revealed that the growth retardation was due to elongation of the G2-M phase, however, the kinase activity associated with Cdc2 remained unaltered. The results indicate that Sam68 may play a critical role in G2-M progression in a manner independent of the control of cyclin/Cdc2 kinase activity.