Separations of bile acids by micro high-performance liquid chromatography (micro-HPLC) using an immobilized 3 alpha-hydroxysteroid dehydrogenase post-column and a spectrofluorimeter are described. The sensitivity of detection is greatly increased by pre-mixing beta-nicotinamide adenine dinucleotide with the mobile phase, using a single pump. The detection limit is 0.04-0.7 ng, depending on the solute. Instruments suitable for the analysis of bile acids in micro-HPLC are discussed.