Abstract Serial samples of bile from the human being and dog were examined for a sulfate conjugate of bilirubin by S 35O 4 tracer studies and the diazotized benzidine colorimetric method. It was demonstrated that the diazotized sulfanilic acid derivatives of bilirubin could be freed completely of S 35 activity. This was accomplished by 2 dimensional paper chromatography or by extracting with organic solvents followed by cold water washing while the azo derivatives were adsorbed to zinc hydroxide gel. A molecular relationship between alkali-stable, direct reacting bilirubin and sulfate could not be demonstrated. The significance of the sulfate moiety present in this fraction is unknown. It is concluded that identification of the alkali-stable, direct reacting fraction of conjugated bilirubin with a nonglucuronide, water-soluble conjugate of bilirubin is not yet justified. Glucuronide glucuronic acid has regularly been associated with the alkali-stable bilirubin azopigment B spot on paper chromatograms. The bilirubin sulfate previously described has been examined in more detail. Better yields are obtained by the use of a much smaller proportion of concentrated sulfuric acid to acetic anhydride. The polar, direct diazo reacting compound has been characterised as a bilirubin sulfonate on the basis of its behavior in various reactions. The most likely site of the conjugation is at the pyrrol nitrogen. The number of sulfonate groups per bilirubin molecule has not been determined. The compound is easily oxidized to biliverdin and reduced to urobilinogen as the sulfonate.