Tritiated commercial bilirubin, mixture of bilirubin isomers was separated into bilirubin Ⅲα, IXα and XⅢα by thin layer chromatography of McDonagh's method and each fraction was further purified by the method. Biliary excretion following intravenous loading of (3)H-bilirubin isomers to Wistar strain rats was serially determined. Compositions of the isomers in the biles collected from Wistar strain rats, heterozygote Gunn rats and normal human subjects were determined. The results obtained were as follows. 1) The excretion ratio in 0-2 hours following the loading of (3)H-bilirubin isomers was 46.0% of (3)H-bilirubin Ⅸα, 45.3% of (3)H-bilirubin Ⅲα and 46.9% of (3)H-bilirubin XⅢα respectively. The biliary excretion within 24 hours following the administration of (3)H-bilirubin isomers was 74.7% of (3)H-bilirubin Ⅸα, 73.3% of (3)H-bilirubin Ⅲα and 75.5% of (3)H-bilirubin XⅢα respectively. No statistically significant difference among the excretion ratios of the isomers in these hours fractions was calculated. (p<0.1) 2) The compositions of biliary (3)H-bilirubin isomers determined by thin layer chromatograms after separate loading of tritiated Ⅲα, Ⅸα and XⅢα in the order were Ⅲα fraction 95.8%, 2.6% and 0.6%, IXα fraction 3.5%, 93.8% and 3.2% and XⅢα fraction 0.7%, 3.6% and 96.2%, respectively. Combined loading of tritiated Ⅲα and XⅢα isomers resulted in the bile composition of Ⅲα 49.1%, IXα 3.6% and XⅢα 47.3%. These results suggest a intact recovery of those isomers. 3) The bile composition of the isomers in unloading Wistar strain rat was Ⅲα 4.59%, IXα 89.83% and XⅢα 5.58%. Contents of Ⅲα and XⅢα of these rats were significantly increased when compared with those loaded with (3)H-IXα isomer. Ⅲα (t=4.56, 0.005＞p＞0.001), XⅢα (t=6.83, 0.001＞p). 4) The composistion of biliary isomers in heterozygote Gunn rats was Ⅲα 4.18%, IXα 91.01% and XⅢα 4.81%. Those in normal subjects was Ⅲα 4.13%, IXα 90.61% and XⅢα 5.26%. According to the results mentioned above, it is considered that a fairly detectable amount of bilirubin Ⅲα and XⅢα exist in the native bile of mammals and that their biliary excretion ratio in the unit hours can be the same level of IXαisomer.