Aim: The two bacterial species, Eubacterium ramulus and Enterococcus casseliflavus, which had previously been isolated from human faeces using the flavonoid quercetin3-glucoside as the growth substrate, were tested for their ability to utilize this compound in vivo. Methods and Results: Germ-free rats were associated with Eu. ramulus and subsequently with Ent. casseliflavus and vice versa. Identification and enumeration of the bacterial cell counts in faeces and intestinal contents were performed by whole cell fluorescence in situ hybridization. Eubacterium ramulus and Ent. casseliflavus occurred in caecal and colonic contents at cell counts of up to 10(10) g(-1) dry weight. In the jejunum, only Ent. casseliflavus was found (10(9) g(-1) dry weight). Upon oral administration of 32 mu mol quercetin3-glucoside, quercetin was detected in the faeces and urine of germ-free rats (2.2 x 10(-1)-8.1 x IO-I mu mol 24-h(-1) faeces collection and 1.0 x 10(-2)-2.8 x 10(-1)mu mol 24-h(-1) urine collection, respectively) and of rats monoassociated with Ent. casseliflavus (7.9 x 10(-1)-2.7 mu mol 24-h(-1) faeces and 1.0 x 10(-1)-5.9 x 10(-1) mu mol 24-h(-1) urine, respectively). In contrast, the faeces and urine of rats associated with Eu. ramulus contained 3,4-dihydroxyphenylacetic acid (4.7 x 10(-2)-3. 6 mu mol 24-h(-1) faeces and 2.4 x 10(-2)-1.0 mu mol 24-h(-1) urine, respectively) but only low, or undetectable, concentrations of faecal quercetin (up to 9.3 x 10(-2) mu mol 24-h(-1) faeces; detection limit 2.5 x 10(-2) mu mol). Urinary quercetin concentrations varied markedly from undetectable amounts up to 1.0 mu mol 24-h(-1) urine (detection limit 1.0 x 10(-2) mu mol). Isorhamnetin was found in the urine of all animals independent of their bacterial status. There were no significant differences between the groups (2.0 x 10(-2)-2.8 x 10(-1) mu mol 24-h(-1) urine). In complete intestinal tissues of animals, associated with. both species, quercetin-3-glucoside and its metabolites were detected by a more sensitive and selective method at concentrations that were two to three orders of magnitude lower than in faeces or-urine. Conclusions: These results indicate that Eu. ramulus may be a key organism for the bacterial transformation of flavonoids in the gut.