The peak of glycyrrhizin (GL) β-D-glucuronidase activity for Ruminococcus sp. PO1-3 and Eubacterium sp. GLH changed to 24 h from 12 h of culture and to 12 h from 48 h, respectively, at almost the same level by the addition of 1.0 mM GL. This enzyme activity was about 20-fold higher in Eubacterium sp. GLH than in Ruminococcus sp. PO1-3. GL β-D-glucuronidase activity of Ruminococcus sp. PO1-3 with Eubacterium sp. GLH and the intestinal flora showed a maximal peak at 12 h of culture in the presence and absence of 1.0 mM GL. This enzyme activity was about 2.5-fold higher in mixed bacteria than in intestinal flora. 3β-Hydrosteroid dehydrogenase activity of Ruminococcus sp. PO1-3 and Ruminococcus sp. PO1-3 with Eubacterium sp. GLH was suppressed greater in the presence of GL than without GL. Also, Ruminococcus sp. PO1-3, Eubacterium sp. GLH, and a mixture of both and intestinal flora, metabolized 1.0 mM GL to glycyrrhetic acid (GA) in yields of about 10, 70, 40 and 100%, respectively, with 24 h culture. From the level of GL β-D-glucuronidase activity, it is considered that the metabolism of GL by intestinal flora is due to both enzymatic and non-enzymatic reactions. Moreover, GA at a concentration of 1.0 mM suppressed growth of Ruminococcus sp. PO1-3, Eubacterium sp. GLH, and the mixture of both and intestinal flora, which metabolized 1.0 mM GA to a negligible amount of 3-oxo-glycyrrhetic acid, indicating the accumulation of unchanged GA. GL β-D-glucuronidase activity of intestinal flora was enhanced by GA, which stimulated bactria possessing particular this characteristic.