Yes belongs to the Src family of protein-tyrosine kinases. In order to understand molecular aspects of its signaling, we decided to isolate proteins that bind to the modulatory region of the Yes molecule. By generating anti-idiotypic antibodies against the aminoterminal domain of the Yes protein, we have identified, characterized and cloned a cDNA for a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. The protein is of 65 kiloDalton (kDa) molecular mass, it is phosphorylated in vivo on serine and is particularly rich in proline. We named it YAP65 for Yes-Associated Protein of 65 kDa. Within the YAP65 sequence, we identified a motif, PVKQPPPLAP, similar to that found in proteins that bind to the SH3 domain of the Abl kinase. Competition assays with synthetic peptides showed the involvement of the predicted proline-rich sequence in binding between YAP65 and the Yes kinase. The YAP65 protein was also shown to bind to other signaling molecules that contain SH3 domains including Nck, Crk and Src. At lower stoichiometry, YAP65 was also shown to bind to the SH3 domains of Abl and guanosine triphosphatase activating protein (GAP). Further characterization of YAP65 should illuminate Yes signaling pathways and could also identify a novel link between protein-tyrosine and serine kinases.