Scr homology 3 (SH3) domain-mediated protein-protein interactions have been implicated in the localization of proteins to specific sites within the cell. We present evidence that the product of the vav proto-oncogene, p95vav, interacts specifically with the focal adhesion protein zyxin both in vitro and in yeast two hybrid system. Solution binding and two-hybrid system experiments demonstrate that association of Vav with the LIM domain protein zyxin is mediated by the C-terminal SH3 domain of the Vav and involves the proline-rich N-terminus of zyxin. The interaction appears to be selective, since no binding of the proline-rich N-terminus of zyxin with other SH3 domain-containing proteins such as GRB-2, phospholipase C gamma, GTPase-activating protein, or p85 was detected.