Serum bilirubin was fractionated by a new reversed-phase "high-performance" liquid-chromatographic (HPLC) procedure, on Micronex RP-30, a polyacryl ester. The five fractions were: 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]. We found close correlation with results of the modified HPLC fractionation of Lauff et al. (J Chromatogr 1981;226:391-402), except for the beta' fraction, which was eluted after beta. The Micronex HPLC involves a simple pretreatment of serum samples, in contrast with the complex preparation described by Lauff et al., and is convenient for routine use in the clinical evaluation of hyperbilirubinemia. We could quantify B delta, BDG, BMG, and unconjugated bilirubin even in sera with normal values for total-bilirubin concentrations. Photoderivatives of bilirubin such as the beta' fraction could be separated and quantified by the same procedure, making the method feasible for pediatric research.