The derivatization of bile acids into trimethylsilyl ether isobutyl ester (IBTMS) and of neutral sterols into trimethylsilyl ether (TMS) allowed the separation on an OV-1 capillary gas chromatography column of 15 bile steroids as follows: cholesterol, 7 alpha-hydroxycholesterol, 6 beta-hydroxycholesterol, 6 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, lithocholate, deoxycholate, 25-hydroxycholesterol, chenodeoxycholate, cholate, murocholate, hyodeoxycholate, ursodeoxycholate, hyocholate, and beta-muricholate. Fragmentation data of the coupled gas chromatographic-mass spectrometric (GC-MS) analysis of these nine bile acids as IBTMS derivatives under electron impact and chemical ionizations (methane, isobutane, and ammonia) are given. The ammonia chemical ionization appears to be the best mode for compound identification and quantitation due to fragmentations into high mass ions. The comparison of methylene units of the five sterols as TMS derivatives and of each type of methyl, TMS, or isobutyl ester of the nine bile acids as TMS ethers showed that isobutyl esterification increased dramatically the retention time of the bile acids, allowing their separation after the neutral sterols. Different methods of GC-MS analysis were applied to the study of bile steroid secretion in long-term rat liver epithelial cell lines, either serum-supplemented cell lines or serum-free cell lines, growing in serum-free medium since the primary explanation or after adaptation of serum-supplemented lines to this medium. It is demonstrated for the first time that liver epithelial cell lines maintain the metabolic pathway leading from synthesized cholesterol to dioxygenated sterols and the two normal main primary bile acids of the liver, chenodeoxycholic acid and cholic acid, up to 32-47% of the in vivo daily rate, and in addition the production of alpha-muricholic acid, the bile acid marker of murine liver.