There have been a few reports of infants with severe neonatal cholestasis related to a defect in primary bile acid synthesis. To assess the importance of such deficiency among children with progressive intrahepatic cholestasis (Byler disease), screening for inborn errors in bile acid synthesis was performed by fast atom bombardment ionization-mass spectrometry of urine samples from 30 affected children. Bile acid analysis revealed a specific fast atom bombardment ionization-mass spectrometry profile for 3 beta-hydroxy-C27 steroid dehydrogenase/isomerase deficiency in five children who had jaundice, hepatosplenomegaly, and fatty stools beginning at ages ranging from 4 to 46 months. None of them had pruritus. Liver function tests showed persistently normal serum gamma-glutamyltransferase activity, low serum cholesterol and vitamin E levels, normal serum bile acid concentrations despite raised serum bilirubin levels, and decreased prothrombin time and clotting factor V. In four of the cases a similar disease was observed in siblings. Liver function returned to normal after oral ursodeoxycholic acid therapy. We conclude that 3 beta-hydroxy-C27-steroid dehydrogenase/isomerase deficiency should be considered when idiopathic cholestatic liver disease with clinical features akin to Byler disease is characterized by the association of normal serum gamma-glutamyltransferase activity, normal serum bile acid concentration, absence of pruritus, and a return to normal liver function during ursodeoxycholic acid therapy. Early identification of these children is essential because they benefit from bile acid therapy and might thus avoid the need for liver transplantation.