In gall bladder bile from 6 male beagles the total C 19O 2 steroid concentration, as measured by mass fragmentography, varied from 3.1 to 5.4 mg/l. More than 95% of the metabolites were present as glucuronide conjugates with the remainder as monosulphates. In the glucuronide fraction, androsterone, aetiocholanolone epiandrosterone, dehydroepiandrosterone, 5β-androstane-3α,17β-diol, 5α-androstane-3β,17α-diol, 5α-androstane-3β,17β-diol and 5-androstene-3β,17β-diol were identified. Quantitatively, the 3α,5β and 3β.5α isomers predominated. In the monosulphate fraction the unsaturated metabolites, 5-androstene-3α,17β-diol, 5-androstene-3β,17α-diol and 5-androstene-3β,17β-diol were relatively more abundant and androsterone and aetiocholanolone were the predominant oxosteroids. The faecal excretion of these steroids in a male beagle, measured over two consecutive 24 h periods, was 157.3 and 194.2 μg/24 h. More than 92% of the steroids were unconjugated. Both the qualitative and quantitative pattern of the C 19O 2 steroids in the unconjugated and glucuronide fractions from faeces were very similar to those found in the biliary glucuronides. All the steroids found as monosulphates in bile were also detected in the monosulphate fraction of faeces. On the second day of a 3 day course of oral ampicillin administration the faecal excretion of C 19O 2 glucuronides was increased from 10.5, under control conditions, to 313.7 μg/24 h. The concentration of unconjugated steroids was unchanged. On the following day the concentration of steroid glucuronides returned to normal but the amounts of unconjugated C 19O 2 metabolites were significantly increased. Ampicillin had no major effect on the relative distribution of steroids measured in either the glucuronide or unconjugated steroid fraction. These results suggest that the biliary-faecal axis is a major excretory route for androgen metabolites in the male beagle. In addition, the effects of ampicillin administration suggest that intestinal microflora play a significant role in the hydrolysis of the biliary steroid glucuronides.