beta-Glucuronidase activity was determined in dialyzed and undialyzed urine from 50 healthy adult males by single-point and enzyme kinetic measuring systems. The enzyme had a pH optimum of 5.2 and a Michaelis constant of 1.465 +/- 0.206 mmol/l (mean +/- SD) of phenolphthalein glucuronide. Substrate inhibition occurred at a concentration of 0.006 mol/l in one fourth of the urine samples. A significant amount of a competitive inhibitor, D-glucaro-1,4-lactone, was present in one third of the specimens. The activity measured by the single-point determination was always lower than the maximal velocity, particularly in the presence of the competitive inhibitor and substrate inhibition. Such pitfalls could be avoided by the enzyme kinetic method which not only permits the detection of substrate inhibition of the enzyme and the elimination of 2-hour dialysis of urine, but also allows the determination of both the maximal velocity of the enzyme and the quantity of D-glucaro-1,4-lactone in urine.