Phenobarbital, by inducing liver microsomal enzymes, may affect bile acid synthesis from cholesterol and thus alter the secretion of biliary lipids and the composition of bile. We, therefore, determined the effects of phenobarbital on bile flow, biliary lipid secretion, bile acid synthesis, and bile-acid pool size. Using an experimental preparation that allows controlled interruption of the enterohepatic circulation (1), we administered 5 mg/kg per day of phenobarbital to healthy Rhesus monkeys for 1-2 wk to achieve steady-state conditions. Three animals were studied with an intact enterohepatic circulation and three with a total bile fistula, each animal served as its own control. Total bile flow and secretion of bile salt, phospholipid, and cholesterol were measured every 24 h during steady-state conditions. Further, under conditions of an intact enterohepatic circulation bile-acid synthetic rate was measured in three animals and pool size estimated in two animals during both control and drug treatment periods. Phenobarbital at doses of 5 mg/kg per day increased bile flow 30-50% in all animals (P < 0.001). The increased bile flow resulted from both an increased "bile-salt independent fraction" and an increased bile-salt secretion rate. Phenobarbital significantly increased bile salt (P < 0.01) and phospholipid secretion (P < 0.05) by about 30% but cholesterol secretion was not significantly changed. Consequently, the concentration of cholesterol relative to bile salt and phospholipid was decreased (P < 0.001). Phenobarbital significantly enhanced the maximal rate of bile acid synthesis 25-30% in all three monkeys with total bile fistulas (P < 0.05) and also augmented bile acid synthesis and pool size in animals with intact enterohepatic circulations despite the fact that the rates of bile salt returning to the liver in these animals would have inhibited bile acid synthesis in control animals. Thus, phenobarbital not only increases the maximal rate of bile acid synthesis but also alters the normal control mechanisms by which bile salts returning to the liver inhibit bile salt synthesis. The fact that phenobarbital treatment results in increased synthesis of bile salt and unchanged secretion of cholesterol is consistant with the view that the drug augments conversion of hepatic cholesterol to bile salt. The resulting decrease in relative cholesterol content in bile may have therapeutic implications for cholesterol gallstone therapy.