The bile was collected from the rat external bile fistula after the duodenal administration of (35)S-sodium sulfate. The exsistence of bilirubin sulfate in the bile was studied. Also clinical significance of bilirubin sulfate was studied useing jaundiced urine obtained from the patients with acute hepatitis, liver cirrhosis and obstructive jaundice. The amount of sulfate was measured by Weber & Schalm's method. The results were as follows: 1) Appearance of (35)S-radioactivity into the rat bile was found from 5 to 6 minutes after the duodenal administration of (35)S-sodium sulfate. The total amount of radioactivity up to 8 hours was 42.1% of given (35)S-radioactivity. 2) Crude direct bilirubin fraction obtained from the rat bile contained 88% of total bile radioactivity. 3) Crude direct bilirubin was fractionated with paper chromatography, then (35)S-radioactivity was dectected with radioautogram and radio chromatogram obtained from the paper chromatogram. The (35)S-radioactivity was found at two spots of Rf 0.32-0.41 and 0.20-0.25. The radioactivity was localized at the original spots of paper chromatogram obtained from the mixture of direct bilirubin and (35)S-sodium sulfate. 4) Amount of bilirubin sulfate decreased at 12 hours when the liver parenchyma was highly damaged after acute carbon tetrachloride poisoning, then increased. 5) No significant corelation was found among the ratio of bilirubin sulfate to direct bilirubin and non-specific colloid reactions, serum transaminases, alkaline phosphatase, total bilirubin and direct bilirubin. 6) Bilirubin sulfate obtained from the jaundiced urine was ranged from 2.09 to 10.0% of the direct bilirubin and averaged 5.98% when bilirubin sulfate was caliculated as bilirubin monosulfate.