Bilirubin conjugates in the serum of cholestatic pediatric patients were investigated with Micronex high-performance liquid chromatography. Serum bilirubin was resolved into four fractions: delta bilirubin (Bd), bilirubin diglucuronide (BDG), bilirubin monoglucuronide (BMG), and unconjugated bilirubin (Bu). The conjugated bilirubin (BDG+BMG) fraction in preoperative patients with biliary atresia (BA) was 48.8 +/- 5.1%, which was significantly higher than that in patients with infantile hepatitis (P < .01). Among postoperative BA patients who recovered from jaundice, the Bd fraction increased during the first month, remained elevated (60% to 80%) for a while, and then gradually decreased. After 6 months, the Bd fraction decreased to 30% in the jaundice-free survivors, but was still higher than that in controls (7%). Even after the total serum bilirubin had normalized (< or = 1.0 mg/dL), distribution of bilirubin fractions remained abnormal, possibly reflecting impaired hepatic excretion of bilirubin. In conclusion, measurement of the conjugated bilirubin fraction enabled BA to be differentiated from infantile hepatitis, and the delta bilirubin fraction proved to be an important indicator of cholestasis in postoperative BA patients with normal serum bilirubin.