Unconjugated and mono- and diconjugated bilirubin was determined by alkaline methanolysis and thin-layer chromatography in serum from 16 pediatric patients with homozygous beta-thalassemia (aged 1-23 years), in 14 age-matched controls, and in 26 healthy adults. Total bilirubin amounted to 21 +/- 20 mumol/L in patients with thalassemia compared with 7 +/- 4 mumol/L in healthy children and 11 +/- 3 mumol/L in healthy adults. The fractions of bilirubin conjugates in the various groups were 8 +/- 4% (patients with thalassemia), 18 +/- 5% (age-matched controls; p less than 0.001), and 16 +/- 5% (healthy adults; p less than 0.001). The low fraction of bilirubin conjugates in patients with thalassemia showed no correlation to any other physical or laboratory finding. It is concluded that subnormal values of bilirubin conjugates in thalassemia indicate defective bilirubin conjugation.