Lithocholic acid, chenodeoxycholic acid and deoxycholic acid but not cholic acid or the glycine or taurine conjugates of these bile acids are glucuronidated by agarose-bound UDPglucuronyltransferase from rt liver in the presence of UDPglucuronic acid. The identification of bile acid glucuronides is based on the specific enzymatic hydrolysis, incorporation of 14C-labeled glucuronic acid into the molecule and by mass spectrometry. Mass spectrometry indicates that bile acid glucuronides formed by agarose-bound UDPglucuronyltransferase are 3-glucuronides. The glucuronidation of lithocholic acid, chenodeoxycholic acid and deoxycholic acid is linear up to 45 min and at a protein concentration between 0.5 and 2.0 mg per assay. The pH optimum of the enzyme reaction is 7.0. The Km for lithocholic acid, chenodeoxycholic acid and deoxycholic acid, which are 0.040, 0.031 and 0.037 mM, respectively, are similar to those found in microsomal preparations. The use of agarose-bound UDP-glucuronyltransferase offers the possibility to synthesize bile acid glucuronides which are needed for kinetic studies in man and animal.