beta-Glucuronidase (EC 3.2.1.31) was purified from human liver and its activity was determined by enzyme kinetic method employing phenolphthalein glucuronic acid (PGA) and conjugated bilirubin, primarily bilirubin diglucuronide purified from human bile, as substrates in the absence or presence of D-glucaro-1,4-lactone. The enzyme was capable of acting on both PGA and conjugated bilirubin with Michaelis constants of 0.435 mmol/l at 56 degrees C and 1.02 mmol/l at 37 degrees C, respectively. Both reactions were beta-glucuronidase-specific because both were inhibited by D-glucaro-1,4-lactone in a competitive fashion. Conjugated bilirubin acted as a noncompetitive inhibitor of the enzyme for PGA in a two-substrate system. The study indicates that these two substrates bind at different catalytic sites of the enzyme and, on molar base, conjugated bilirubin had higher affinity for the enzyme and less degree of inhibition by D-glucaro-1,4-lactone than PGA. Whether such catalytic sites are also common for other beta-D-glucuronid ethers and esters remains to be proven.