A rat liver epithelial cell line growing in a serum-supplemented medium expressed biosynthetic pathways of bile sterols and of free and conjugated chenodeoxycholic and cholic acids, the main primary bile acids of the liver. They were identified and measured by gas chromatography-mass spectrometry. The bile steroid secretion in the serum-supplemented cell line was established upon incubation in a serum-free medium which was demonstrated to sustain cell growth, allowing elimination of the interference of exogenous bile steroids and effectors. The free bile acid secretion was also expressed in a subline adapted to proliferate in this serum-free medium, i.e., a basal medium supplemented with 4 g/l albumin carrying 7.6 muequiv./l of a mixture of six long-chain free fatty acids but without any addition of hormones and growth factors. In addition, the rat liver epithelial cell line growing in the serum-supplemented medium maintained, with time, a steady-state of bile acid secretion over a lifespan of 500 days. In the two types of liver epithelial cell lines, dexamethasone and chenodeoxycholic acid supplementation exerted, individually, either a stimulating or an inhibiting effect on the bile acid secretion concurrently with the hydroxylation of chenodeoxycholic acid into alpha-muricholic acid.