Frozen sections of theratstomach were incubated with 125I-labeled porcine secretin, and then secretin binding sites were localized by autoradiography. Saturable binding was observed only in the muscularis externa (circular and longitudinal smooth muscle layers) of the proximal nonglandularforestomach. Saturable binding was quantitated by densitometry. 125I-porcine secretin bound to a single class of high-affinity binding sites with a dissociation constant of 0.6 nM. Porcine andratsecretins were nearly equipotent in inhibiting saturable 125I-porcine secretin binding, and vasoactive intestinal polypeptide, peptide histidine-isoleucine, and glucagon were much weaker. Carbachol (100 microM) stimulated a sustained increase in tension inforestomachmuscle in vitro, and porcine secretin causedrelaxationof this stimulated contraction. We conclude thatratforestomachsmooth muscle expresses a high-affinity specific secretin binding site that mediatesrelaxation. This putativesecretin receptormay mediate some of the actions of secretin on gastric motility.