The present study was designed to investigate whether the metabolism of the opiate analgesic alfentanil in humans is subject to the debrisoquine 4-hydroxylation polymorphism. The role of a specific cytochrome P-450 form, debrisoquine 4-hydroxylase, in the metabolism of alfentanil was investigated by competitive inhibition experiments over the concentration range 4-100 microM. Alfentanil was incubated with human liver microsomes in the presence of an NADPH-generating system. Alfentanil and its major metabolites were quantified in the incubates by reversed phase high-performance liquid chromatography (HPLC). Alfentanil was rapidly metabolized, yielding noralfentanil as the main metabolite. Kinetically, alfentanil metabolism occurred monophasically and the kinetic parameters were 22.8 microM for Km app and 3.86 nmol alfentanil metabolized min-1.mg protein-1 for Vm app. Debrisoquine was a weak, noncompetitive inhibitor of alfentanil metabolism and of the formation of its major metabolites, with Ki values between 2.00 and 3.21 mM. It can be concluded that alfentanil is not metabolized in vitro by the human cytochrome P-450 form involved in debrisoquine 4-hydroxylation; therefore, the in vivo disposition of the drug is most likely not affected by deficiency of this enzyme.