Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.