We used a highly specific method, alkaline methanolysis-high performance liquid chromatography, for determining the concentration and patterns of the unconjugated and esterified bilirubin fractions in the sera of pediatric patients with hepatobiliary disease. Bilirubin-protein conjugates were assayed using a new method that selectively removes bilirubin reversibly bound to protein, allowing measurement of the tightly bound bilirubin-protein conjugates by use of a diazo method. Fifty-two serum samples from children with varying bilirubin concentrations and diagnoses were studied. Whereas no conjugated pigment was detectable in the serum samples of healthy children or in individuals with Gilbert syndrome or Crigler-Najjar syndrome, bilirubin monoester and diester conjugates and bilirubin-protein conjugates were present in the sera of children with cholestatic liver disease, and accounted for 69% +/- 15% of the total bilirubin in these samples. Bilirubin fractional analysis was incapable of differentiating extrahepatic biliary obstruction from hepatocellular disease, because of overlap between the groups. The presence of bilirubin-protein conjugates in serum always coincided with detection of bilirubin monoester and diester conjugates. The distribution of bilirubin and its conjugates in sera provides a sensitive, although nonspecific, measure of hepatic disease.