We describe an accurate reverse-phase high-performance liquid chromatography (HPLC) method for the separation and quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in faeces and intestinal contents from germ-free (GF) rats. We demonstrated that female GF rats excreted predominantly BMG and that the percentage of this conjugate was at most 71.7% of the total bilirubin excreted with the faeces. The highest percentages for BDG and the UCB were 27.9% and 6.0%, respectively. The bile pigment composition in duodenal contents was 59.8% BDG and 40.2% BMG (median percentage) and was 47.7% BDG, 50.1% BMG and 2.2% UCB in ileal contents. Deconjugation of BDG to BMG was profound in caecal contents with 26.0% BDG, 67.4% BMG and 6.6% UCB. Endogenous (mammalian) beta-glucuronidase activity was present in intestinal contents throughout the entire length of the intestine and in faeces of the GF rats. The results indicated that it is very likely that endogenous beta-glucuronidase plays a role in the deconjugation of bilirubin glucuronides as well as of other glucuronides in the intestine of the GF rat.