The mobilities of several free and conjugated 5 beta-bile acids, cholesterol and analogues, and alpha, beta-unsaturated sterols and steroidal acids have been investigated with a microbore reversed-phase high-performance liquid chromatographic column (50 cm X 1 mm I.D., 12% C18) with appropriate solvent mixtures at flow-rates of 50-100 microliter/min and a UV monitor set at 193, 198, 212, or 243 nm. With a solvent mixture of 2-propanol-10 mM phosphate buffer, pH 7.0 (160:340) bile acids or their conjugates were separated in a manner similar to those by microBondapak columns (10% C18). The lower detection limit of the conjugates was 20 pmoles with the UV detector set at 193 nm, whereas the lower limit for alpha, beta-unsaturated keto sterols or steroidal acids was 5 pmoles at 243 nm. The detection limit for cholesterol with the UV monitor at 198 nm was 10 pmoles. Contributions of substituent groups of sterols to their time of elution (capacity factor) were calculated for several substituted 4-cholesten-3-ones.