Non bilirubin fraction which was extracted from bile of various liver and biliary tract diseases and prepared from crystalline bilirubin after irradiation by fluorescence was studied. Following results about the nature of non bilirubin fraction positive for diazo reaction and its clinical significance was obtained. 1) The material obtained in the fraction was yellowish and soluble in water and showed positive diazo reaction. Its absorption maximum showed 545-555nm in water solution (pH 2.4) and unchanged by acidulation. 2) In thin layer chromatogram of this azo pigment of non bilirubin fraction, two to four spots appeared usually. This results suggested that it was not composed from one substance. The Rf values were different from those of already reported azo pigments in thin layer chromatogram and it was confirmed that the Rf value of one of these spots was changed by methylation. 3) Percentage of non bilirubin fraction in total bilirubin in bile of various liver and biliary tract diseases was significantly increased in comparison with that of control subjects. This ratio increased in the order of the group of constitutional jaundice, cirrhosis of the liver and chronic hepatitis respectively. 4) Significant correlations between percentage of non bilirubin fraction in total bilirubin and liver function tests (ZnTT, TTT, γ-globulin) were obtained. These findings suggested that non bilirubin fraction was increased in the case of hepatic parenchymal damage or the disturbance of the bilirubin conjugation enzyme.