In gas chromatographic-mass spectroscopic analysis of buprenorphine metabolites, the urine specimen must be first hydrolyzed to release buprenorphine and norbuprenorphine from their glucuronide conjugates. For evaluation of existing hydrolysis methods and to find out the optimal hydrolysis conditions, buprenorphine-3-beta-D-glucuronide (B3G) and norbuprenorphine-3-beta-D-glucuronide (NB3G) were synthesized. In a previous communication we reported on the optimum conditions for hydrolysis of B3G. Because we have previously shown that no hydrolysis was achieved under basic conditions and that acid hydrolysis resulted in extensive degradation, only enzymatic hydrolysis was examined for NB3G. This study therefore reports on the optimum enzymatic conditions for the hydrolysis of NB3G. Urine fortified with synthetic NB3G was hydrolyzed with beta-glucuronidases from different source species, including Helix pomatia, Escherichia coli, and Patella vulgata. Glusulase, a preparation containing both the beta-glucuronidase (H. pomatia) and sulfatase, was also tested. It was found that marked differences exist in the reactivity of these enzymes. Incubation with glucuronidases from H. pomatia or E. coli at 37 degrees C for 16 h resulted in quantitative hydrolysis of NB3G. At 60 degrees C, complete hydrolysis was achieved with 1000 units of H. pomatia in 4 h and after only 1 h with glusulase. On the other hand, incubation at 60 degrees C for 4 h with Patella vulgata resulted in only 14.7% hydrolysis.