A high-performance liquid chromatographic method with fluorescence detection has been developed for the determination of human faecal bile acids, especially free faecal bile acids. Faecal bile acids were extracted using an Amberlite XAD-2 column and then fractionated into four groups (free, glycine-conjugated, taurine-conjugated and sulphated bile acids) on a piperidinohydroxypropyl Sephadex LH-20 column. The free bile acid fraction and free bile acids obtained after enzymatic hydrolysis and/or solvolysis of the three other fractions were derivatized with 1-bromoacetylpyrene and dicyclohexyl-18-crown-6-ether. The derivatized bile acids were separated stepwise on a Shim-pack CLC-ODS column using acetonitrilemethanol-water (100:50:30) (A), (100:50:20) (B), and (100:50:0) (C) as mobile phases with changing automatically from A to C using a solvent changer. Calibration curves of bile acid standards were linear in the range between 20 and 400 pmole, when monitored at 370 nm (excitation) and 440 nm (emission). Percent recoveries of bile acids from human faecal samples were between 80 to 95%. The method is applicable to clinical use and is sensitive, reliable, and useful for the detailed determination of human faecal bile acids, especially of free faecal bile acids.