Analysis of serum unconjugated and conjugated bilirubin fractions by routine diazo procedures does not allow a definite diagnosis of Gilbert's syndrome. By the alkaline methanolysis procedure of Blanckaert followed by thin-layer chromatography we were able to discriminate Gilbert's syndrome even in the presence of normal serum bilirubin concentrations from healthy subjects, patients with chronic persistant hepatitis and patients with chronic hemolysis. The relative proportion of unconjugated bilirubin in serum was 95 +/- 2% in patients with Gilbert's syndrome (n = 28), 84 +/- 5% in healthy subjects (n = 29), 75 +/- 6% in patients with chronic persistant hepatitis (n = 7) and 85 +/- 3% in patients with chronic hemolysis (n = 9). The difference between Gilbert's syndrome and the control groups with normal or elevated serum bilirubin was highly significant (p less than 0.001). In Gilbert's syndrome, unconjugated bilirubin ranged between 90 and 99%, in healthy subjects between 72 and 90%, in patients with chronic persistant hepatitis between 68 and 85% and in patients with chronic hemolysis between 81 and 89% of total. An overlap was only seen in one patient with Gilbert's syndrome and in 2 healthy subjects at the 90% level. We conclude that in most patients with Gilbert's syndrome provocation tests are no longer necessary.