The content of conjugated bilirubin (CB) was about the same in small intestinal contents from germfree (GF) and conventional (CONV) rats. Caecal contents from GF rats contained considerably more CB than from CONV rats. The results indicated that the caecum is the major site of microbial deconjugation of CB in the CONV rat. Separation of azopigments prepared from small intestinal contents of GF and CONV rats revealed similar patterns. Similar separation patterns were also observed with azopigments prepared from caecal contents and faeces of GF rats. Some chemical properties of CB prepared from GF rat faeces were studied.