Alfentanil, sufentanil, and fentanyl are synthetic opioids that are metabolized by oxidative N-dealkylation in the liver. We have previously shown that cytochrome P-450 3A4 (CYP3A4) contributes significantly to human liver microsomal alfentanil oxidation. Since identification of specific drug-metabolizing enzymes allows prediction of the variables affecting drug metabolism, the purpose of the present study was to identify the P-450 enzymes responsible for sufentanil and fentanyl metabolism in human liver microsomes. Microsomal preparations fortified with a reduced nicotinamide-adenine dinucleotide phosphate-generating system were incubated with 0.25 microM 3H-fentanyl or 3H-sufentanil. Rates of N-dealkylated metabolite formation significantly correlated with nifedipine oxidation activity (a marker of CYP3A4 activity) for fentanyl and sufentanil (r = 0.93 and 0.87, n = 18, respectively), but not with the oxidation activity for ethoxyresorufin (CYP1A2), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), or chlorzoxazone (CYP2E1). Gestodene and troleandomycin (chemical inhibitors of CYP3A4) and antibody to CYP3A4 inhibited N-dealkylation of fentanyl and sufentanil. Chemical inhibitors of CYP2C, 2E1, and 2D6 did not inhibit N-dealkylation of fentanyl and sufentanil. Recombinant CYP3A4 expressed in Escherichia coli showed N-dealkylation activity of fentanyl and sufentanil, while expressed CYP1A2, 2C10, and 2E1 enzymes did not. We conclude that CYP3A4 is responsible for fentanyl and sufentanil N-dealkylation in vitro.