We have isolated cDNA clones encoding the rodent 14-3-3 zeta and epsilon isoforms by screening bacteriophage expression libraries with a probe derived from the carboxy-terminus of the Vav oncoprotein. These isoforms, however, did not recognize the full length Vav protein under physiological conditions. In agreement with previous studies (see D Morrison, Science 266, 56-57, 1994), these 14-3-3 proteins bound very efficiently to Raf. The interaction between 14-3-3 zeta and Raf involves the central region of 14-3-3 zeta which includes a motif related to annexins. 14-3-3 zeta binds to Raf independently of Ras and forms stable ternary complexes with these two molecules. In contrast to published reports, we have observed that the catalytic activity of Raf was not activated in Raf/14-3-3 zeta immunocomplexes. Likewise, purified preparations of 14-3-3 zeta had no effect on the kinase activity of Raf immunoprecipitates. In addition, Ras activated Raf regardless of whether it was bound or not to 14-3-3 zeta. Finally, overexpression of 14-3-3 zeta cDNA clones in NIH3T3 cells did not result in detectable morphologic transformation even when co-transfected with plasmids encoding Raf and/or Ras proteins. These observations argue against a critical regulatory role of the 14-3-3 proteins in the Raf mitogenic pathway.