SH3 domains constitute a family of protein-protein interaction modules that bind to peptides displaying an X-proline-X-X-proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non-receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage-displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross-linking, we demonstrate that a proline-X-X-aspartate-tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8-related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3-containing proteins that do not bind to canonical XPXXP-containing peptides, and that establish distinct interactions in the signaling network.