Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF 1 receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF 1 receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30 -41(cCRF 30 -41 ), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an ␣-helical conformation when bound to the ECD. Residues of cCRF 30 -41 comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met 38 and Ile 41 ) as well as two amide groups (Asn 34 and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of nonpeptide antagonists targeting the ECD of this receptor.Corticotropin-releasing factor 1 (CRF 1 ) 2 receptor plays a crucial modulatory role in the central stress response, making it an attractive target for the development of therapeutics for a variety of stress-related disorders, including anxiety and depression (1-5). The CRF 1 receptor is a class B G-protein-coupled receptor (GPCR) and is activated endogenously in mammals by the peptide agonists CRF (6) and the urocortins (7-9). Binding of the native ligands to the receptor promotes a conformational change that leads to G-protein activation inside the cell. Signaling is mediated primarily through the second messenger cAMP via G␣ s ; however, coupling of the receptor to G␣ i and G␣ q/11 has been observed (10, 11). The receptor is expressed both in the periphery, where pituitary activation underlies the major role of CRF activation of the hypothalamic pituitary adrenal axis during stress, and in brain, where extrahypothalamic CRF activates central receptors to mediate mood and affect (12).A two-site binding and activation model has been proposed for agonist binding to class B GPCRs (13). Recent pharmacological data suggest that activation of the CRF 1 receptor is initiated by the C terminus (ϳ30 amino acids) of the peptide ligand interacting with relatively moderate affinity to the extracellular N-terminal domain (ECD) of the CRF 1 receptor (14). This primary interaction has the effect of increasing the local concentration of the ligand and positions its N terminus (ϳ10 amino acids) in the optimal orientation...