The protein-tyrosine kinase encoded by the human c-fes protooncogene (p93c-fes) plays a direct role in myeloid differentiation, but downstream substrates for this kinase have not been identified. Here we report that the human ras GTPase-activating protein (GAP) is a substrate for p93c-fes in vitro. Purified, recombinant GAP was readily phosphorylated on tyrosine residues by bacterially-expressed p93c-fes. Two-dimensional tryptic mapping revealed a single GAP phosphopeptide, consistent with specific phosphorylation of GAP by p93c-fes on one or several closely-spaced tyrosine residues. Autophosphorylated p93c-fes also formed a stable complex with GAP. Complex formation is likely to involve the src homology 2 (SH2) domains of GAP and autophosphorylated tyrosine residues of p93c-fes, as deletion of the fes SH2 domain did not abolish complex formation. Furthermore, immobilized recombinant fusion proteins containing either or both of the GAP SH2 domains were able to precipitate p93c-fes with an affinity equal to that observed with a monoclonal antibody against the recombinant fes protein. Fusion proteins containing the GAP N-terminal, C-terminal catalytic, or SH3 domains did not bind to p93c-fes. Interaction of the GAP SH2 domains with p93c-fes is phosphorylation-dependent, as the recombinant SH2 domain proteins were unable to bind to a kinase-defective c-fes mutant and showed reduced binding of a mutant in which one of the two tyrosine autophosphorylation sites was replaced with phenylalanine. Stimulation of c-fes autophosphorylation in vivo may induce interaction with GAP, resulting in altered p21ras function.