The effect of cholestyramine administration on the enterohepatic circulation of bile acids was studied in eight normal volunteers. In six subjects the metabolism of sodium taurocholate-(14)C was determined after its intravenous injection before and during the 6th wk of cholestyramine administration, 16 g/day. In two subjects, the metabolism of cholic acid-(14)C was observed before and during the 2nd wk of cholestyramine, 16 g/day. Bile acid sequestration resulted in a more rapid disappearance of the injected primary bile acid and its metabolic products. The composition of fasting bile acids was promptly altered by cholestyramine to predominantly glycine-conjugated trihydroxy bile acid. In four subjects, unconjugated bile acid-(14)C was administered during cholestyramine administration; the relative proportion of glycine-conjugated bile acid-(14)C before enterohepatic circulation was similar to the relative proportion of unlabeled glycine-conjugated bile acid present in duodenal contents after an overnight fast, indicating that a hepatic mechanism was responsible for the elevated ratios of glycine- to taurine-conjugated bile acid (G: T ratios) observed. The relative proportions of both dihydroxy bile acids, chenodeoxycholic and deoxycholic, were significantly reduced. Steatorrhea did not occur, and the total bile acid pool size determined after an overnight fast was unaltered by cholestyramine. These findings suggest that in normal man bile acid sequestered from the enterohepatic circulation by cholestyramine is replaced by an increase in hepatic synthesis primarily via the pathway leading to production of glycocholic acid.