Unconjugated bilirubin and its mono- and diester conjugates were measured by alkaline methanolysis and normal-phase high-performance liquid chromatography (AMHPLC) in 195 serum specimens obtained from 63 patients with various hepatobiliary disorders and from 47 healthy adult controls. With this assay, esterified bilirubins were undetectable in the controls, and detection of esterified pigment in a sample was interpreted as an abnormal result. Using this criterion, the AMHPLC result in the clinical anicteric patients (n = 39) was more frequently abnormal (87%) than the corresponding value of fasting serum bile acids (48%), SGPT (52%), total bilirubins (55%), alkaline phosphatase (71%) or gamma-glutamyl transpeptidase (71%). The cumulative frequency of abnormality of these tests was comparable to that of an abnormal AMHPLC result alone. All icteric patients had detectable esterified bilirubins as well as an increased alkaline phosphatase level, while a normal result was found for serum bile acids in 34%, for SGPT in 29% and for gamma-glutamyl transpeptidase in 11%, respectively. In most hyperbilirubinemic patients, total serum bilirubin levels, as determined by a conventional diazo method, exceeded the value obtained by AMHPLC. This discrepancy, which appears to reflect the presence of bilirubin covalently bound to serum protein, was particularly pronounced following desobstructive intervention in patients with obstructive jaundice, in whom the decline of serum bilirubins showed a fast and a slow disappearance component. The latter portion seemed to correspond with slow plasma clearance of bilirubin covalently linked to serum albumin, disappearing at a rate comparable to that of the albumin moiety.