Two novel $eta$-glucuronidases, which metabolize glycyrrhizin (GL) to 18$eta$-glycyrrhetinic acid-3-O-$eta$-D-glucuronide (GAMG), were purified from Streptococcus LJ-22 isolated from human intestinal microflora. $eta$-Glucuronidases I and II were purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, butyl toyopearl, Q-Sepharose, hydroxyapatite Ultrogel, and GL-attached Sepharose column chromatographies, with the final specific activities of 137 and 190 nmole/min/mg, respectively. The molecular sizes of both $eta$-glucuronidases were found to be 140 kDa by gel filtration, and they consisted of two identical subunits (M.W. 67 kDa by SDS-PAGE). $eta$-Glucuronidases I and II showed optimal activity at pH 7.0 and pH 6.5, respectively. Both purified enzymes were potently inhibited by $Cu^{2+}$ and PCMS, and had maximum activity on glycyrrhizin, but did not hydrolyze p-nitrophenyl-$eta$-glucuronides, baicalin, or GAMG These findings suggest that the biochemical properties and substrate specificities of these enzymes are different from those of the previously purified $eta$-glucuronidases. This is the first reported purification of sugar (not aglycone)-recognizing $eta$-glucuronidases from intestinal bacteria.