Src homology 2 and 3 (SH2 and SH3) domains mediate protein-protein interactions in intracellular signaling by protein-tyrosine kinases (PTKs). We have isolated cDNA clones from mouse embryo cDNA expression library that encode a new signaling protein which we call Efs (Embryonal Fyn-associated Substrate). The deduced amino acid sequence of 560 residues in length revealed one SH3 domain at its amino-terminal region, two proline-rich motifs with the consensus sequences of binding to Src-family SH3s, and a cluster of YXXP motifs that are possibly tyrosine-phosphorylated to serve as ligands binding to SH2 domains. Structure and alignment of these characteristics sequences are homologous to those of p130Cas, but Efs and p130Cas are different proteins. Expression of the Efs gene was higher in placenta, embryo and brain than in other adult tissues. Transfection of COS-7 cells with a plasmid encoding an epitope-tagged Efs resulted in the expression of a 83 kDa protein. The epitope-tagged Efs was hyperphosphorylated when cotransfected with a vector expressing Fyn. In an in vitro kinase assay with the PCC4 cell lysate, Efs became phosphorylated on tyrosine residues and coprecipitated with p59fyn and p62yes; the result suggests that Efs is a physiological substrate of these PTKs.