Most digested food 2 h after overnight feeding in rat remained in the stomach, duodenum, upper small intestine, lower small intestine, cecum and colon, all of which indicated pH between 4 and 7 and had glycyrrhizin (GL) hydrolyzing activity. This enzyme activity was highest in the cecal and colonic contents among all gastrointestinal contents. Also, 3α-hydroxyglycyrrhetic acid (3α-hydroxyGA) and 3β-hydroxyglycyrrhetic acid (3β-hydroxyGA) oxidizing enzymes were localized in the same cecal content. Namely, rat gastrointestinal bacteria had the ability to hydrolyze GL to 3β-hydroxyGA by glycyrrizin β-D-glucuronidase and to oxide 3β-hydroxyGA and 3α-hydroxyGA to 3-oxoGA by 3β-hydroxyglycyrrhetinate dehydrogenase and 3α-hydroxyglycyrrhetinate dehydrogenase, respectively. In medium of pH 1 to pH 10, metabolites 3β-hydroxyGA, 3-oxoGA and 3α-hydroxyGA obtained from the metabolism of GL were the highest in pH 8. The intestinal contents of pH 6 or pH 7 were able to produce metabolites 3β-hydroxyGA in the metabolism of GL. However, the stomach content at pH 4.2 was lowest in metabolite 3β-hydroxyGA. It is unknown whether or not GL is metabolized to 3β-hydroxyGA by the stomach content in vivo.