Tritiated commercial bilirubin, mixture of bilirubin isomers was separated into bilirubin IIIα, IXα and XIII αby thin layer chromatography of McDonagh's method and each fraction was further purified by the method. Biliary excretion following intravenous loading of 3H-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 3H-bilirubin isomers was 46.0% of 3H-bilirubin IXα, 45.3% of 3H-bilirubin IIIα and 46.9% of 3H-bilirubin XIIIα respectively. The biliary excretion within 24 hours following the administration of 3H-bilirubin isomers was 74.7% of 3H-bilirubin IXα, 73.3% of 3H-bilirubin IIIα and 75.5% of 3H-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 3H-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 3H-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 IXα isomer.