Non-bilirubin fraction was extracted from the bile obtained from Wister strain rats and heterozygote Gunn rats, and separated by Ostrow(1)s method with minor modification. Azo-pigments was prepared with the non-bilirubin fraction after addition of Ehrlich(1)s diazo reagent. Significance of non-bilirubin fraction showing positive diazo reaction (NBAPF) was studied on the basis of the relation between the percentage of NBAPF to total bile azo pigments (proportion of NBAPF) and molar ratio of glucuronic acid to the ester-form bilirubin. The following results were obtained; 1) Averaged proportions of NBAPF were 0.94% in Wister strain rats and 2.50% in heterozygote Gunn rats, respectively. The latter proportion was higher than the former but not significant due to the wide variation of the measured values of the latter group. 2) Good correlation between the proportion of NBAPF and the molar ratio of glucuronic acid to the ester-form bilirubin was observed. 3) No significant correlation was observed between the bilirubin concentration in the bile and the molar ratio of glucuronic acid to the ester-form bilirubin or the proportion of NBAPF. 4) These results suggest when the conjugation of bilirubin and glucuronic acid in the liver was impaired, both the excretion of dipyrryl substance yielded from bilirubin and other conjugation mechanism increase compensatory.