An orthodox diazo method is popularly used for measuring bilirubin. On the other hand, an enzymatic method which employs bilirubin oxidase, has also been in use for considerable time. We have often found disparities between direct bilirubin values obtained with the enzymatic and the diazo methods. To determine the cause of these disparities, bilirubin subfractions were analysed and classified into two types by HPLC. Samples showing great differences contained conjugated, unconjugated and delta bilirubins (type I), while samples showing only small differences contained almost exclusively unconjugated bilirubin and delta bilirubin (type II). Conjugated bilirubin is therefore largely responsible for the differences observed between the two methods. Particularly marked differences were found for bile (in which all the bilirubin is conjugated) and for synthetic conjugated bilirubin. Bilirubin oxidase decreases the absorbance at 450 nm when it catalyses oxidation of bilirubin, but after the oxidation of synthetic conjugated bilirubin at pH 3.7 another peak appeared at 450 nm, as shown by HPLC and spectrophotometry, but not when the reaction was performed at pH 7.2, namely under conditions permitting complete oxidation. Incomplete oxidation products of conjugated bilirubin are responsible for the disparity. Care is therefore needed in the clinical interpretation of direct bilirubin values measured by the enzymatic method.