Saturable binding sites for 125I-Bolton-Hunter substance P were observed in frozen sections of the oxyntic mucosa of the canine stomach using quantitative autoradiography. The cell type possessing substance P binding sites in this region was identified as the chief cell in 2 ways. First, the saturable binding of radioiodinated substance P correlated with chief cell content (and not with parietal cell content, for example) in dispersed oxyntic mucosal cells fractionated by centrifugal elutriation. Second, saturable binding of radioiodinated substance P was localized to dispersed chief cells by autoradiography using emulsion-coated preparations of isolated cells affixed to glass slides. Parietal and mucous cells did not bind substance P. In studies of enriched chief cell preparations, the binding of radiolabeled substance P was found to be time- and cell number-dependent, specific, saturable, reversible, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent Kd of 105 pM and a Bmax of 3000 receptors per cell. In competitive displacement studies, the order of potency of analogs for inhibition of the saturable binding of radiolabeled substance P to chief cells was substance P = physalaemin greater than substance K greater than neuromedin K; thus, the chief cell has a substance P-preferring tachykinin binding site. Bombesin, cholecystokinin, and somatostatin had no effect on substance P binding. Substance P stimulated pepsinogen secretion from isolated canine oxyntic glands in dose-dependent fashion with a half-maximal response occurring at a substance P dose of about 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)