Fasting levels of cholic acid conjugates were determined by radioimmunoassay in the serum of 24 patients with extensively documented Gilbert's syndrome and in 98 healthy controls without unconjugated hyperbilirubinemia. The Gilbert's syndrome patients studied included all three subtypes, as determined from studies of the plasma disappearance kinetics of sulfobromophthalein and indocyanine green. Although patients with structural liver disease severe enough to produce hyperbilirubinemia almost invariably have elevated fasting serum levels of cholic acid conjugates, values were normal in each of the Gilbert's syndrome patients, including patients with metabolic abnormalities in sulfobromophthalein and indocyanine green transport. It is concluded that the measurement of fasting serum levels of cholic acid conjugates is a useful adjunct to the diagnosis of Gilbert's syndrome.