The microsomal metabolism of fentanyl, a synthetic opioid commonly used in anesthesia, was investigated in human liver. Incubation of fentanyl with human hepatic microsomes fortified with NADPH resulted in the formation of a single major metabolite, namely norfentanyl, as determined by GC/MS. No evidence was obtained for the formation of either desproprionylfentanyl or N-phenylpropionamide, the latter arising via N-dealkylation of the fentanyl amide nitrogen. Kinetic analysis of microsomal fentanyl oxidation revealed a single K(m) of 117 microM and a Vmax of 3.86 nmol of norfentanyl formed/min/nmol of cytochrome P450 (P450). Studies using chemical inhibitors of human P450 enzymes revealed that only agents known to inhibit CYP3A4 (e.g. ketoconazole and erythromycin) were capable of strongly inhibiting (> or = 90%) microsomal fentanyl oxidation. Marked inhibition (> 90%) of norfentanyl formation by liver microsomes was also observed with polyclonal antibodies to CYP3A4, whereas antibodies to other human P450s were without effect. Furthermore, rates of norfentanyl production by 10 individual human liver samples were highly correlated (r2 = 0.876, F = 56.46 p < 0.001) with immunochemically determined levels of CYP3A4 present in the samples but not with levels of CYP2C8, CYP2C9, CYP2C19, or CYP2E1. Our results indicate that CYP3A4 is the major catalyst involved in fentanyl oxidation to norfentanyl in human liver. Alterations in CYP3A4 levels or activity, as well as the concomitant administration of other therapeutic agents metabolized by this P450 enzyme, could lead to marked perturbations in fentanyl disposition and, hence, analgesic response.