Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand‐ and receptor‐expressing cells. Consequently, the ephrins and ephrin receptor tyrosine kinases often mediate processes involving cell–cell contact, including cellular adhesion or repulsion, developmental remodeling and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small‐cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and is suitable for co‐crystallization studies. Complex structures show multiple contacts between kinase and substrates; in particular, two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes.