Calcitonin gene-related peptide (CGRP) is an endogenous vasodilator peptide that produces its effects by activation of CGRP1 and CGRP2 receptor subtypes. These receptor subtypes are characterized in functional studies using the agonist Cys(Acm)2, 7-human-alpha-calcitonin gene-related peptide (Cys(ACM)2, 7-h-alpha-CGRP), which activates CGRP2 receptors, and the antagonist h-alphaCGRP(8-37) which has a high affinity for CGRP1 receptors and a low affinity for CGRP2 receptors. Our aim was to identify factors that may limit the use of these drugs to characterize CGRP receptor subtypes. We studied CGRP receptors using isolated ring segments of pig coronary and basilar arteries studied in vitro. The affinity of the antagonist h-alphaCGRP(8-37) for inhibiting h-alphaCGRP-induced relaxation of coronary arteries (log10 of the antagonist equilibrium dissociation constant = -5.33) was determined from Schild plots that had steep slopes. Therefore, we used capsaicin to investigate the role of endogenous CGRP in confounding affinity measurements for h-alphaCGRP(8-37). After capsaicin treatment, the slopes of the Schild plots were not different from one, and a higher affinity of h-CGRP(8-37) in blocking relaxation was obtained (log10 of the antagonist equilibrium dissociation constant = -6.01). We also investigated the agonist activity of the putative CGRP2 receptor selective agonist Cys(Acm)2,7-h-alphaCGRP. We found that maximal relaxation of coronary arteries caused by Cys(Acm)2,7-h-alphaCGRP was dependent upon the level of contractile tone induced by KCl. We also determined the KA for Cys(Acm)2,7-h-alphaCGRP and found that the KA (817 nM) was not significantly different from the EC50 (503 nM) for this drug in causing relaxation, indicating that Cys(Acm)2, 7-h-alphaCGRP is a partial agonist. Because experimental conditions affect the actions of h-CGRP(8-37) and Cys(Acm)2,7-h-alphaCGRP, the conditions must be carefully controlled to reliably identify CGRP receptor subtypes.