Background . No information is available on the hepatic and extrahepatic pathways of bile acid synthesis in normal children and in pediatric cholestatic liver diseases. Methods .To explore the changes of the two pathways of bile acid synthesis during development, plasma concentrations of 7α-hydroxycholesterol and 27-hydroxycholesterol were measured in 50 healthy children (1 month–14 years) and compared to 18 adult controls. We also measured plasma oxysterols in 31 patients with pediatric cholestatic liver disease. Results . A progressive increase of plasma concentrations of both 27-hydroxycholesterol and 7α-hydroxycholesterol was found with age. In children with cystic fibrosis-associated liver disease plasma concentrations of 27-hydroxycholesterol were significantly lower compared to age-matched controls (5.6 ± 0.5 vs. 12.8 ± 1.1 μg/dl; p