The metabolism of 14C-labeled chenodeoxycholic, cholic, and deoxycholic acids was studied in patients with intrahepatic cholestasis. Radioactively labeled metabolites were isolated from urine and were identified by gas-liquid chromatography-mass spectrometry. About 5% of the radioactivity was recovered in urine after administration of labeled chenodeoxycholic acid to a patient with mild intrahepatic cholestasis. In urine collected 0-24 hr after the injection, 20% of the radioactivity appeared in the combined glycine and taurine conjugate fractions, and the predominant metabolite in these fractions was identified as hyocholic acid. Eighty percent of the activity was eluted in the sulfate fraction presumably representing mainly sulfated chenodeoxycholic acid conjugates. Twenty percent of the radioactivity was recovered in urine following administration of labeled cholic acid to a patient with biliary cirrhosis and severe cholestasis. In urine collected on the fifth day, half of this radioactivity appeared in the glycine and taurine conjugate fractions, and 10% of this activity was present as tetrahydroxycholanoates. The major metabolites in this fraction were 3 alpha, 6 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta- and 1 xi, 3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholanoic acids. The former compound constituted about 50% of the tetrahydroxycholanoates. Three additional minor tetrahydroxy bile acids were present, one of which was tentatively identified as 6 beta-hydroxycholic acid. About 5% of the radioactivity appeared in urine after oral administration of labeled deoxycholic acid to a patient with mild intrahepatic cholestasis. Twenty-two percent of the activity appeared in the glycine and taurine conjugate fractions isolated from urine collected on the second day after the administration. About 75% of this activity was associated with trihydroxycholanoates. The main metabolite was 1 beta-hydroxydeoxycholic acid with small amounts of, tentatively, 6 alpha-hydroxydeoxycholic acid.