Norfentanyl has been identified previously as a urinary metabolite of fentanyl. However, at clinically relevant concentrations, norfentanyl are below the limits of detection. The use of labeled drug in metabolic studies is a standard approach to overcome the limitations imposed by metabolite concentrations that are below detection limits. Unfortunately, the available tritium-labeled fentanyl yields unlabeled norfentanyl following N-dealkylation. Thus, we have developed a technique to monitor the N-dealkylation of fentanyl using the other products of N-dealkylation. The biotransformation of fentanyl was studied in human liver microsomes. After incubation with human liver microsomes for 20 min, almost 50% of a 0.03 microM concentration of [3H]-fentanyl was metabolized to the [3H]N-dealkylated metabolite phenylacetaldehyde, which was then converted principally to [3H]2-phenylethanol and to a smaller extent to [3H]phenylacetic acid in microsomal incubates. The apparent Km,app and Vmax,app for norfentanyl formation were 82 +/- 21 microM and 4.7 +/- 0.4 nmol product formed/min/nmol cytochrome P450, respectively. Thus, this study defined methodology that can be used to evaluate the metabolism of fentanyl, both in vivo and in vitro, at clinically relevant concentrations.