Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp(105) of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser(108) and its epitope may reside within residues 105-132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand-receptor interaction.