Basal secretion of gastrin by dog antral mucosa incubated in air, at 37 °C, in phosphate-buffered Robinson's solution, pH 7.4, containing 5.5 mM glucose decreased sharply after a 10-min incubation period. Gastrin release proceeded unimpaired for prolonged periods of time when the incubation medium was renewed at 10-min intervals. The marked decrease of gastrin secretion was not the result of a negative feedback of gastrin on its own release since increasing concentrations (50-200 pg/ml) of exogenous hormone had no effect on the rate of antral gastrin secretion. One hour incubation solutions derived from dog antral tissues exerted a rapid inhibitory effect on gastrin release by fresh canine antral mucosa. The inhibitory medium did not interfere with the radioimmunoassay of gastrin. Exogenous gastrin 17 was recovered quantitatively following incubation with inhibitory media, suggesting that the marked decrease in gastrin accumulation did not result from metabolic degradation of the hormone by proteolytic enzymes leaking into the bathing solutions. Withdrawal of the inhibitory solutions and addition of fresh buffer resulted in prompt resumption of basal and acetylcholine (50mM) stimulated antral gastrin secretion. The inhibitory action on gastrin release was restricted to incubation media derived from gastrointestinal tissues. Collectively these results suggest the presence of an inhibitor in the incubation solutions of canine gastrointestinal tissues which rapidly and reversibly suppresses antral gastrin release.