Despite many reports that serotonin (5-HT) inhibits gastric acid output, the role and mechanism of action of endogenous 5-HT to modulate gastric secretion remain unclear. Vagal stimulation enhanced the basal rate of 5-HT release into both the gastric lumen (600%) and the portal circulation (265%) of the rat. The peak rate of 5-HT release into the portal circulation was 1,000-fold higher that luminal release (12 μg/min and 1.2 ng/min, respectively). To elucidate site(s) of action of 5-HT to inhibit acid secretion, several approaches were taken. Intraluminal perfusion of exogenous 5-HT to encompass enhanced levels seen after vagal stimulation did not reduce gastric acid output. In contrast, administration of systemic 5-HT, which raised portal venous 5-HT to similar levels as vagal stimulation, had a marked antisecretory effect. Chemical or surgical ablation of enteric or sympathetic nerves innervating the stomach did not attenuate the inhibitory effect of exogenous 5-HT on gastric acid output. The antisecretory effect of systemic 5-HT was insensitive to pretreatment with piroxicam, doxantrazole, close gastric intra-arterial sodium nitroprusside, somatostatin monoclonal antibody, or bilateral adrenalectomy. The results suggest that 5-HT is released from endogenous stores into the portal circulation in sufficient quantities after vagal stimulation to alter gastric physiology and that its action is independent of the autonomic nervous system, gastric mucosal prostaglandins or somatostatin, mucosal mast cell or adrenal constituents, or changes in gastric mucosal blood flow.