The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of Hck.GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (delta CAT) or the Src homology region (delta SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, delta CAT, and delta SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.