The physiological significance of serotonin released into the intestinal lumen for the regulation of motility is unknown in humans. The aim of this study was to evaluate the effect of serotonin infused into the lumen of the gastric antrum, duodenum or the jejunum, on antro-duodeno-jejunal contractility in healthy human volunteers. Manometric recordings were obtained and the effects of either a standard meal, continuous intravenous infusion of serotonin (20 nmol/kg/min) or intraluminal bolus infusions of graded doses of serotonin (2.5, 25 or 250 nmol) were compared. In addition, platelet-depleted plasma levels of serotonin, blood pressure, heart rate and electrocardiogram were evaluated. All subjects showed similar results. Intravenous serotonin increased migrating motor complex phase III frequency 3-fold and migrating velocity 2-fold. Intraluminal infusion of serotonin did not change contractile activity. Platelet-depleted-plasma levels of serotonin increased 2-fold following both intravenous and high doses of intraluminal infusions of serotonin. All subjects reported minor short-lived adverse effects following intravenous serotonin stimulation, while only half of the subjects reported minor short-lived adverse effects following intraluminal serotonin stimulations. We conclude that exogenous serotonin in the lumen of the upper part of the small intestine does not seem to change antro-duodeno-jejunal contractility significantly in healthy adult volunteers.