The CRF receptors are members of a 7-transmembrane receptor family that includes GH-releasing hormone (GRF), calcitonin, vasoactive intestinal peptide (VIP), secretin, and PTH receptors. To determine the structural features of the CRF receptor that may influence ligand recognition, a series of mutant receptors was analyzed for binding to astressin, a CRF antagonist, and to urocortin, a CRF agonist. Mutant receptors included chimeras between the CRF-R1 and GRF-R or Activin IIB-R, a single membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using 125I-[DTyr1] astressin (Ast*) and 125I-[Tyr0]-rat urocortin (Ucn*). There was no binding to a chimeric receptor in which the first extracellular domain (E1c) (i.e. the N-terminal region) of the CRF-R1 was replaced by that of the GRF-R. The complementary chimera in which E1 domain of the GRF-R was replaced by that of the CRF-R1 bound astressin and urocortin with Ki values approximately 10 nM, compared with inhibitory binding dissociation constant (Ki) values of approximately 2-4 nM for the wild-type CRF-R1. The chimera in which E1 of the activin IIB receptor was replaced by E1 of the CRF-R1 bound astressin with a Ki approximately 4 nM. A chimera in which both the first and fourth extracellular domains of the CRF-R1 replaced the corresponding domains of the GRF-R bound astressin with Ki approximately 4 nM and urocortin with a Ki approximately 2 nM. A chimera in which all four extracellular domains of the CRF receptor replaced those of the GRF-R bound astressin and urocortin with Ki values approximately 4 nM and approximately 1 nM, respectively. In conclusion, the major determinants for high affinity binding of CRF agonists and antagonists to CRF-R1 are found in the first extracellular domain of the receptor.