A recently developed reverse-phase high-performance liquid chromatography procedure was applied to the analysis of bilirubins present in serum of neonates. In normal newborns, unconjugated (UCB) and conjugated bilirubins (CB) rose in parallel from day 2 to day 4: the average conjugated fraction remained between 1.2 and 1.6% of total pigment, significantly below the 3.6% documented in adults. However, the absolute concentration of conjugates was 2- to 6-fold higher than the serum levels in adults. Bilirubin conjugates were detected in all samples, but only 20% of these were bilirubin diconjugates, in contrast to a mean of 54% in the adult population. The increased absolute concentrations of UCB and CB together with the decreased fractional amount of conjugates and with the decreased diconjugates seem to be the result of an increased bilirubin production combined with a relative defective biliary secretion. Serum bile acids were high but did not correlate with the levels of bilirubin conjugates. The pattern of serum bilirubins was not different in 4 newborns with hemolytic syndromes. Cholestasis occurred in another child with hemolysis; this was evident from the high concentration and the high fractional amount of conjugates and from the high serum bile acids level.