14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.