An enzymatic bilirubin assay using bilirubin oxidase was evaluated. Its repeatability and linearity were excellent, and excellent correlations were observed between the enzyme and the diazo methods in total and conjugated bilirubin. The coefficient of correlation between the conjugated bilirubin fraction obtained with the diazo method and that of HPLC was 0.424, while this coefficient was 0.580 for the diazo method. A multi-laboratory study using 3 serum specimens revealed lower coefficients of variation for the enzymatic method than for the diazo method. Bilirubinuria specimen showed good accord between total bilirubin and conjugate bilirubin levels for the enzymatic method, not for the diazo method. When 12 newborn baby specimens and 34 pediatric specimens were studied, all exhibited increased concentrations of conjugated bilirubin with the diazo method. In an adult case with prolonged jaundice, the diazo method usually yielded higher conjugated bilirubin values than the enzymatic method. The enzymatic method was then concluded to be superior to the diazo method for conjugated bilirubin determination.