Glycyrrhizin (GL) was hydrolyzed to glycyrrhetic acid (GA), glycyrrhetic acid mono-beta-D-glucuronide (GAMG) or both by glucuronidases in various organs of rat. GL beta-D-glucuronidase I, hydrolyzing GL to GA; GAMG beta-D-glucuronidase, hydrolyzing GAMG to GA; and 3alpha-hydroxyglycyrrhetinate (3alpha-hydroxyGA) dehydrogenase, oxidizing 3alpha-hydroxyGA to 3-oxo-GA were found in the organs of this animal. GL beta-D-glucuronidase II was distributed in the lysosomal fraction of all organs except brain; 3alpha-hydroxyGA dehydrogenase was distributed in the microsomal fraction of the liver; but other enzymes were distributed in the nuclear, lysosomal, microsomal and soluble fractions of a variety of organs. GL beta-D-glucuronidase I, GL beta-D-glucuronidase II and GAMG beta-D-glucuronidase activities in a mixture of lysosomes and microsomes of rat liver exhibited different patterns on hydroxyapatite column chromatography. These results showed the metabolic pathways of GL to be of two types: a beta-D-glucuronidase hydrolyzing GL to GA, and the other consisting of two different beta-D-glucuronidases hydrolyzing GL to GAMG and GAMG to GA.