Raf-1 is a serine/threonine protein kinase that connects cell surface receptor signals to nuclear transcription factors. By screening Schizosaccharomyces pombe (S. pombe) cDNA library, we isolated Rad24, which is a 14-3-3 homolog that is important in the DNA damage checkpoint in S. pombe, as a Raf-1 interacting protein. The interaction found in yeast was confirmed by co-immunoprecipitation. Furthermore, Cdc25, which has been known to bind to Rad24, also associated with Raf-1 and was phosphorylated in vitro by catalytically active Raf-1. However, in the presence of Raf-1, an interaction between Rad24 and Cdc25 was inhibited in triple hybrid assay, indicating that Raf-1 inhibits the interaction between Rad24 and Cdc25. An in vitro competition assay showed that the binding of Cdc25 and of Rad24 to Raf-1 is mutually exclusive. Western blots of whole cell lysates probed with polyclonal antibodies specific for tyrosine-15-phosphorylated Cdc2 showed that overproduction of Rad24 led to the dephosphorylation of tyrosine residue on Cdc2, which is known to be activated through dephosphorylation by Cdc25 phosphatase. Unexpectedly, overexpression of catalytically inactive mutant protein of Raf-1, S624A, also caused tyrosine dephosphorylation of Cdc2. Thus, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 or a direct phosphorylation of Cdc25, bypassing the checkpoint pathway in DNA repair through Cdc25 activation.