Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor α (hTGF-α) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-α and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg45 in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-α with high affinity (KD) = 13-21 and 35-40 nM, respectively), sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu367, Gly441, or Glu472 to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-α and were recognized by Mab528. However, mutant Gly441Lys showed markedly reduced binding to hEGF, implicating Gly441, in the L2 domain, as part of the binding site that recognizes Arg45 of hEGF.