The similarity of the catalytic domains of Raf and Src family members suggests that functions of homologous residues may be similar in both kinase families. A tryptophan residue, W260, in the WEI region of the Src family kinase Hck has an important role in regulating ATP binding. We tested the hypothesis that the tryptophan, W342, in the WEI region of c-Raf may have a similar role to the W260 of Hck. Mutation of W260 to A in Hck activates kinase activity, but we found that mutation of W342 to A in c-Raf inactivates the kinase activity. Mutating W342 to aspartate (D), lysine (K) or histidine (H) also inactivated c-Raf whether assayed as a purified immunoprecipitate or when recruited to the plasma membrane. A constitutively active c-Raf can be generated by mutating two regulatory tyrosines to aspartate. When placed into this active c-Raf mutant, mutation of W342 to D, K or H enabled phosphorylation and activation of the c-Raf substrate MEK at the plasma membrane but not in an immunoprecipitation assay. We conclude that (1) Tryptophan has a different role in the WEI regions of c-Raf and Hck, (2) W342 is not directly involved in MEK binding as both positive and negative residues at 342 are permissive for MEK activation at the membrane in a constitutively active c-Raf mutant, (3) Factors at the membrane are capable of potentiating activation of c-Raf containing mutated W342 in a hyperactivated c-Raf, but not in a wild type c-Raf and (4) There is a stringent structural requirement for W at residue 342 in c-Raf.