Long-term studies were performed on dogs previously prepared by cholecystectomy, ligation of the lesser pancreatic duct, and insertion of a duodenal cannula. After an overnight fast, bile duct cannulation and stabilization of bile flow with intravenous (IV) sodium taurocholate, serotonin, 10 micrograms/kg/min, or 0.15 N NaCl was infused. In similar experiments, animals were fed a standard meal, and serotonin or 0.15 N NaCl was infused IV beginning simultaneously with or 30 minutes after the meal. Short-term experiments were performed on dogs anesthetized with pentobarbital and prepared by abdominal evisceration, cholecystectomy, and bile duct cannulation. Serotonin caused significant inhibition of fasting bile formation (3.8 +/- 0.3 ml/15 min to 3.2 +/- 0.3 ml/15 min), meal-stimulated choleresis (4.0 +/- 0.3 ml/15 min to 3.5 +/- 0.3 ml/15 min), and bile flow in eviscerated animals (1.6 +/- 0.1 ml/15 min to 1.1 +/- 0.2 ml/15 min). Bile acid output and 14C erythritol clearance were stable while bile bicarbonate output was decreased during serotonin infusion. A similar inhibitory effect was demonstrated with serotonin, 5 micrograms/kg/min, but the inhibition was not statistically significant with 2.5 micrograms/kg/min. These studies demonstrate that serotonin inhibits bile acid-independent bile formation, possibly at the ductular level, and the inhibition occurs independently of endogenous gastrointestinal tract hormone secretion.