A novel high-performance liquid chromatography (HPLC) system is described for the separation of bilirubin and its conjugates in body fluids. Biliary bilirubin mono- and diglucuronide can be analysed directly on a C18 reversed-phase column with acetonitrile-dimethyl sulphoxide-0.1 M ammonium acetate (pH 5.16) (50:50:85, v/v/v) as mobile phase. However, the simultaneous determination of conjugated and unconjugated bilirubins in plasma required conversion of the conjugates into their methyl esters by alkaline methanolysis before HPLC separation of the C18 column eluted with acetonitrile-dimethyl sulphoxide-0.50 M ammonium acetate (pH 4.6) (50:50:40, v/v/v). The method is superior to, and more flexible than, previously described reversed-phase systems by allowing precise control of retention times by adjustment of pH, buffer concentration and the relative proportion of organic modifier in the mobile phase.