It has been previously reported that treatment of urinary oxazepam by commercial β-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid-liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant--approximately 1% (0.59-1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.