Immunoreactive plasma motilin concentrations were studied following a variety of stimuli in 24 healthy fasting subjects. Plasma motilin was measured by a radioimmunoassay using antibody GP 71 (J. C. Brown) and natural porcine motilin as standard. Basal motilin levels ranged from undetectable to 365 pg/mL. Antral and intraduodenal infusion of 50 mL 0.1 N HCl (pH 1.2) at 5 mL/min failed to alter significantly plasma motilin levels but duodenal acid infusions at 17 mL/min caused a significant increase (70.8 ± 29.5 pg/mL, mean ± SEM; n = 6), maximal at 40 min. Duodenal alkalinization with 50 mL 0.3 M Tris buffer (pH 8.0) infused at 5 mL/min produced no change in plasma motilin. A mixed meal did not affect plasma motilin levels. Ingestion of 60 g fat significantly increased plasma motilin (n = 13; maximal increase 150.3 ± 43.3 pg/mL at 30 min) but duodenal infusions of fat failed to increase plasma motilin levels. These results suggest that motilin secretion induced by fat requires that the fat be present initially within the stomach for secretion to occur. We conclude that ingested fat is a potent stimulus of motilin release. As duodenal acidification (50 mL, 0.1 N HCl over 10 min) induces duodenal activity resembling migrating motor complexes but does not release motilin, our data argue against the release of motilin following duodenal acidification as a trigger for the initiation of these complexes in man.