Glucagon increases hepatocellular cAMP and decreases biliary cholesterol output. In these experiments, we examined the relation between cAMP and biliary cholesterol secretion. Bile flow and composition were measured in conscious dogs previously prepared by cholecystectomy, ligation of the lesser pancreatic duct, and placement of duodenal and gastric cannulae. Sodium taurocholate (500 mg/hr) was given intravenously to stabilize bile flow. After 2 hr of taurocholate infusion, dibutyryl cyclic AMP (160 mg kg-1 hr-1) or theophylline (20 mg kg-1 hr-1) was administered intravenously. Dibutyryl cAMP caused a decrease in both cholesterol concentration (242 +/- 25 micrograms/ml to 81 +/- 11 micrograms/ml) and cholesterol output (692 +/- 102 micrograms/15 min to 382 +/- 47 micrograms/15 min). Theophylline decreased cholesterol concentration (282 +/- 39 micrograms/ml to 221 +/- 21 micrograms/ml), but there was no significant change in cholesterol output. Bile flow increased significantly with both dibutyryl cAMP (2.8 +/- 0.2 ml/15 min to 4.9 +/- 0.2 ml/15 min) and theophylline (2.6 +/- 0.4 ml/15 min to 4.2 +/- 0.4 ml/15 min). In additional experiments, aminophylline (85% theophylline, 15% ethylenediamine) was administered intravenously (24.7 mg kg-1 hr-1). Aminophylline reduced cholesterol concentration (59 +/- 6 micrograms/ml to 36 +/- 5 micrograms/ml), but cholesterol output was stable. Bile flow increased significantly (3.7 +/- 0.2 ml/15 min to 6.5 +/- 0.4 ml/15 min). The mechanisms of these changes remain unknown. The effect of dibutyryl cAMP on biliary cholesterol secretion supports but does not prove the hypothesis that glucagon decreases biliary cholesterol output via the second messenger, cAMP.