The benzodiazepines are among the most widely prescribed drugs in clinical medicine because of their extensive use as anxiolytics, sedative-hypnotics, anticonvulsants, and muscle relaxants. Reports on the B-glucuronidase hydrolysis of conjugated benzodiazepines have accumulated in the last few years; however, there is no quantitative kinetic data on hydrolysis of the different benzodiazepine-glucuronides with B-glucuronidase. Since differences may exist when the enzyme hydrolyzes these structurally different benzodiazepine conjugates, it is important that the rate of hydrolysis of different substrates catalyzed by B-glucuronidase be understood. This study found that in 50 mM phosphate buffer pH 6.4, lorazepam-glucuronide has the highest turnover rate of 3.7 s (exponent -1) with an associated Km of about 100 mM, followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s (exponent -1) with an associated Km of 60 mM. Temazepam-glucuronide has the lowest rate of 0.94 s (exponent -1) with an associated Km of 34 mM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for B-glucuronidase was determined to be between 6 and 7 when the enzyme hydrolyzes the benzodiazepine conjugates in buffered drug-free urine. Effects of temperature and incubation time were also examined. The electron donating or withdrawing of the individual benzodiazepine structure may play an important role in the reactivity of the lorazepam-glucuronide, oxazepam-glucuronide, and temazepam-glucuronide catalyzed by B-glucuronidase. This is consistent with other observations made for monosubstituted phenyl-B-glucuronides by Wang et al. 2 tables, 5 figures, and 16 references