Recently we isolated Rad24, a 14-3-3 homologue, which is essential for DNA damage checkpoint, as a Raf-1 interacting protein by screening a Schizosaccharomyces pombe (S. pombe) cDNA library. Raf-1 was also found to recognize Cdc25 that is sequestered and inactivated by Rad24. In the present study, experiments were performed to determine the effect of overexpression of Raf-1 proteins on asynchronously growing S. pombe cells. The overexpression of Rad24 induced elongated cell morphology and reduction in growth rate, resulting in cell cycle arrest while the overexpression of catalytically active Raf-1 led to a decrease in cell size at division in S. pombe. However, the active Raf-1 failed to rescue the growth arrest induced by Rad24 overexpression. In addition, the cells carrying catalytically active Raf-1 were significantly more radiosensitive than those from a normal control as assessed by ultraviolet sensitivity assay, suggesting that constitutive overproduction of Raf-1 kinase can revert DNA replication checkpoint arrest caused by UV irradiation. Taken together, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 in DNA repair, bypassing the checkpoint pathway through Cdc25 activation.