Glucuronidation of drugs represents a major pathway of human drug metabolism. Numerous studies show that the glucuronides formed can accumulate during chronic therapy and/or have direct pharmacological activity. In both cases, cleavage of the glucuronide by human beta-glucuronidase (beta-Gluc) would release the parent compound, thereby modifying drug disposition. Variability in expression of beta-Gluc could therefore be a confounding factor for interindividual variability in drug disposition both in the setting of accumulating glucuronides or for the use of glucuronides as prodrugs, such as the nontoxic glucuronide-spacer derivative of doxorubicin (Dox-S-G). We therefore investigated expression and function of beta-Gluc in human liver (n = 30) and human kidney (n = 18). Cleavage of the model compound 4-methylumbelliferyl-beta-D-glucuronide (MUG) revealed a wide range of activities in liver (0.32-1.85 mumol/mg/h, mean value 0.87 +/- 0.34 mumol/mg/h) and kidney (0.07-1.00 mumol/mg/h, mean 0.39 +/- 0.21 mumol/mg/h), which followed a log normal distribution. Variable enzyme activity was closely correlated to enzyme expression as assessed by Western blotting (r = 0.80, P < .001 and r = 0.71, P < .05 for liver and kidney, respectively). Glycyrrhizin (Ki = 470 and 570 microM), estradiol 3-glucuronide (Ki = 0.9 and 1.2 mM) and paracetamol glucuronide (Ki = 1.6 and 2 mM) were found to inhibit beta-Gluc activity competitively in liver and kidney, respectively. Enzyme kinetics were investigated in detail for MUG and Dox-S-G. Whereas MUG followed monophasic Michaelis-Menten kinetics in liver (K(m) = 1.32 +/- 0.25 mM, Vmax = 1201 +/- 462 nmol/mg/h, n = 3) and kidney (K(m) = 1.04 +/- 0.05 mM, Vmax = 521 +/- 267 nmol/mg/h, n = 3), cleavage of Dox-S-G was best described by the Hill equation, which indicated a cooperative substrate binding pattern of Dox-S-G. In summary, beta-Gluc function shows wide interindividual variability in human liver and kidney that is due to different steady-state levels of the enzyme. Moreover, enzyme kinetics are substrate-dependent, with Dox-S-G showing a cooperative binding. These data indicate the possibility of wide interindividual variability in beta-Gluc-mediated cleavage of drug glucuronides in the human.