Rat calcitonin gene-related peptide (rat CGRP) and related peptides did not cause contraction of gastric smooth muscle cells; however, preincubation with rat CGRP or human CGRP inhibited smooth muscle contraction caused by carbachol. Rat CGRP and human CGRP were equipotent in opposing contraction with a half-maximal effect produced by 0.1 nM, but rat calcitonin-adjacent peptide (rat CAP) and human calcitonin had no effect. Rat CGRP caused an increase in cellular adenosine 3',5'-cyclic monophosphate (cAMP) of 60%, which was augmented to 155% with 1 mM isobutyl methylxanthine (IBMX). IBMX did not influence the sensitivity of the muscle cells to rat CGRP to increase cAMP, the half maximal effect being produced by 0.4 nM in the presence or absence of IBMX. Rat CGRP and human CGRP were equipotent for stimulating cAMP, but rat CAP and human calcitonin had no effect. Binding of 125I-CGRP was temperature dependent, saturable, reversible, and specific. Rat CGRP and human CGRP were equipotent at inhibiting binding of 125I-CGRP and rat CAP, and human calcitonin did not inhibit binding. For rat CGRP and human CGRP, the dose-response curves for binding of 125I-CGRP were broad, and both peptides accelerated the rate of dissociation of bound 125I-CGRP. Computer analysis of dose-response curves demonstrated two classes of binding sites for CGRP, one with a high affinity (Kd 3 nM) and another with low affinity (Kd 3,700 nM). Our results indicate that CGRP interacts with previously undescribed receptors on gastric smooth muscle cells to increase cellular cAMP and inhibit contraction.