The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the nonpeptide antagonist.