The secretion of pepsinogen and intrinsic factor (IF) in response to various known stimulators and inhibitors of gastric acid secretion was examined in isolated rabbit gastric mucosa maintained in organ culture. Acetylcholine (10(-8) M) stimulated stimulated both pepsinogen (P less than 0.01) and IF (P less than 0.01) secretion and this stimulation was blocked by atropine. Parasympatholytic agents did not alter unstimulated (basal) secretion of pepsinogen even at high concentrations (atropine, 10(-2) M or propanthelene bromide, 5 X 10(-3) M), however, at these concentrations basal IF secretion was abolished. Histamine (10(-4) and 10(-2) M) had no effect on pepsinogen secretion but stimulated IF secretion (P less than 0.001). Antagonism of H2 receptors by cimetidine reduced both basal and histamine-stimulated IF secretion, but pepsinogen secretion remained unaltered. Under the conditions of the above experiments the gastric mucosal surface was not exposed to HCl but was constantly buffered by culture medium at pH 7.4. When we applied 50 mN HCl to the mucosal surface of the biopsies, pepsinogen secretion doubled (P less than 0.001) but IF secretion was abolished. These studies have clearly documented that: (1) fundamental differences exist in the responses of pepsin and IF secreting cells; (2) H+ ions bathing the mucosal surface of the stomach may influence the results of experiments designed to examine the mechanisms of gastric mucosal macromolecular secretion.