A method for the simultaneous determination of sulfated bile acids in human bile without prior hydrolysis and solvolysis is described. The sulfate fraction was obtained from a bile specimen by passing it through a Sep-Pak C18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution into the 3-sulfates of unconjugated, glycine- and taurine-conjugated ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate was attained by high-performance liquid chromatography (HPLC) on an SC-02 column. Separation of these sulfates was effected when acetonitrile-0.5% ammonium carbonate (8:31, 8:26 and 8:23, v/v) was used as mobile phase. The sulfated bile acids in human bile were unequivocally identified on the basis of their behaviour in HPLC using mobile phases of various PH values. The present method proved to be applicable to the characterization and quantitation of sulfated bile acids in human bile.