The radioligand binding characteristics and in vitro biological activities of human calcitonin gene-related peptide (hCGRP) homologs, analogs and fragments have been tested in a variety of preparations in order to determine the structural requirements of the calcitonin gene-related peptide (CGRP) receptor. The ligand selectivity profile observed in this study suggests that [125I] hCGRP binding sites present in rat brain and spleen are similar. The high affinity of the analogs [Cys(ACM)2,7]hCGRP and cyclo2,7[Asp2,Lys7]hCGRP indicates that the disulfide bridge between positions 2 and 7 is not essential for the binding of the ligand to the CGRP receptor. On the other hand, the lack of affinity of various N-terminal fragments in the binding assays demonstrates the importance of amino acids 9-37 in maintaining appropriate receptor recognition. The existence of CGRP receptor subtypes is supported by data obtained in various in vitro bioassays. For example, the linear analog, [Cys(AMC)2,7]hCGRP which retained high potency in the rat vas deferens bioassay displayed no agonistic activity in the right and left guinea pig atrial preparations. Additionally, the C-terminal fragment hCGRP12-37 possessed some antagonist properties in atrial but not vas deferens preparations. Thus, it appears that it is possible to differentiate between CGRP receptor subtypes on the basis of differential agonistic (e.g. [Cys(ACM)2,7]hCGRP) and antagonistic (e.g., hCGRP12-37) potencies in various isolated tissues. Moreover, the weak but significant antagonistic properties of hCGRP12-37 may be most useful in order to design more potent CGRP receptor antagonists.