The quantitatively most important conjugated bile acids in man were separated by reversed-phase ion-pair chromatography without prior derivatization. As non-polar matrix an Ultrasphere I.P. column (C 18) was used, and an ionic alkyl compound, tetrabutylammonium phosphate, was added to the mobile phase, which was a mixture of acetonitrile and water. Under these conditions, the glycine and taurine conjugates of cholic acid, chenodcoxycholic acid and deoxycholic acid were separated within 15 min. At 214 nm, the minimum measurable concentration was 1.3 2.0 nmol/ml. The average recovery from bile was 94%. In ten patients with biliary drainage an average of 79.5% of the bile acids were glycine conjugates.