Interleukin-5 receptor alpha is a therapeutic target for hypereosinophilic diseases including allergic inflammations and asthma. The cyclic peptide AF17121 (Ac-VDE[CWRIIASHTWFC]AEE-CONH(2)) has been identified as a submicromolar inhibitor of interleukin 5 (IL5)-interleukin 5 receptor alpha (IL5Ralpha) interaction from a random peptide screen. However, this inhibitor has limitations as a drug lead because of its relatively large size. We used chemical synthesis of peptides with natural and non-natural amino acids along with kinetic binding and cell proliferation competition assays to expand definition of structural elements in the peptide that are important for receptor antagonism and to elucidate the underlying pharmacophore. We found that the specific steric array of hydrogen bonding groups in the Arg 6 guanido side chain is critical for receptor inhibition. We also investigated noncharged structural elements in AF17121. Screening a set of five hydrophobic residues showed that peptide function is strongly sensitive to variations in several of these residues, most prominently Ile 7 and Trp 13. We postulate that presentation of charged, hydrogen bonding and hydrophobic structural elements within the disulfide-constrained peptide drives IL5Ralpha recruitment by AF17121. We hypothesize from these results and previous receptor mutagenesis studies that Arg 6 recruitment of IL5Ralpha occurs through hydrogen bonding as well as charge-charge interactions with Asp 55 in site one of domain 1 of IL5Ralpha, and that this interaction is complemented by additional charged and hydrophobic interactions around the Asp 55 locus. Scaffolding a limited set of structural elements in the inhibitor pharmacophore may be useful for small molecule antagonist design inspired by the peptide.