The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.