Serum Bilirubin was fractionated by newly developed reversed phase high performance liquid chromatography (HPLC) into 5 fractions: delta (delta-Bilirubin, B delta), gamma (bilirubin diglucuronide, BDG), beta (Bilirubin monoglucuronide, BMG), beta' ((Z, E,)- and/or (E, Z)-bilirubin IX alpha) and alpha ((Z, Z)-bilirubin IX alpha). Sera of healthy subjects and of patients with unconjugated hyperbilirubinemia showed predominantly alpha fraction with a small amount of beta' fraction. Trace amounts of delta fraction were detected in a few cases. The results of fractionation of serum bilirubin in 159 patients with various hepatobiliary diseases suggested that the ratios B delta/(B delta + BDG + BMG) and BMG/B delta can be useful parameters to follow patients with jaundice, compared with the reported B delta/total bilirubin which did not always reflect the jaundice stage, especially in cases with low serum bilirubin levels.