Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.