Characterization of the non-glucuronide conjugated bilirubin fraction showing positive phosphate-ester reaction reported by Kondo was studied. The results were as follows: 1) After (32)P-sodium phosphate was injected into the duodenum of Wister strain rat, bile was collected from the external bile fistula. Crude bilirubin was prepared from the collected bile by Sakamoto's method. (32)P-radioactivity was localized at the ester-form bilirubin fraction of the crude bilirubin. (32)P-radioactivity was detected in the bile during from 10 to 15 minutes after the duodenal injection. Total amount of the excreted (32)P-radioactivity collected up to eight hours after the loading was 1.7%. 2) Localization of the radioactivity at radioautogram and radio chromatogram obtained from the paper chromatogram of the ester-form bilirubin fraction separated by Kondo's method was found at the same spot showing positive phosphate-ester reaction reported by Kondo. The radioactivity of the azo-pigments yielded from the ester-form bilirubin localized at the spots showing positive phosphate-ester reaction. 3) Amount of bilirubin phosphate fraction increased in the rat bile after carbon tetrachloride poisoning. Amount of bilirubin phosphate fraction in the bile collected from the patients with liver cirrhosis was much more than that from normal subject. This results suggests the bilirubin phosphate fraction increased compensatly when the bilirubin glucuronide fraction was decreased in the liver parenchymal damage.