Fentanyl, sufentanil, and alfentanil are commonly used as opioid analgesics. Alfentanil clearance has previously been shown to exhibit an important interindividual variability, which was not observed for fentanyl or sufentanil. Differences in pharmacokinetic parameters of alfentanil have previously been associated with the wide distribution of CYP3A4, the only known hepatic cytochrome P450 monooxygenase (CYP) involved in the conversion of alfentanil to noralfentanil. Little is known about the involvement of CYP enzymes in the oxidative metabolism of fentanyl and sufentanil. Microsomes prepared from different human liver samples were compared for their abilities to metabolize fentanyl, sufentanil and alfentanil, and it was found that disappearance of the three substrates was well correlated with immunoreactive CYP3A4 contents but not with other CYPs, including CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP2E1. Specific known inhibitors of CYP enzymes gave similar results, whereas the use of recombinant human CYP enzymes expressed in yeast provided information about the possible involvement of other CYPs than CYP3A4 in the biotransformation of fentanyl and sufentanil. The possible in vivo interaction of fentanyl and sufentanil with other drugs catalyzed by CYP3A4 is also discussed.