Human liver microsomal cytochrome P450s (P450s or CYP) involved in the oxidative biotransformation of the anesthetic agent propofol were investigated. Of six cDNA-expressed human P450 enzymes tested, CYP2B6 and CYP1A2, followed by CYP3A4, had high catalytic activities at a 20 microM propofol concentration, corresponding to clinical plasma levels. K(m) and k(cat) values for propofol omega- and 4-hydroxyation were 27 microM and 21 nmol omega-hydroxypropofol formed/min/nmol CYP2B6 and 30 microM and 42 nmol 4-hydroxypropofol formed/min/nmol CYP2B6, respectively. CYP2B6 expressed in HepG2 cells also effectively catalyzed propofol omega- and 4-hydroxylation. In a panel of individual human liver microsomes, propofol omega- and 4-hydroxylation activities (at the substrate concentration of 20 microM) were highly correlated with CYP2B6 contents, and moderately with CYP3A4 contents. Anti-CYP2B6 antibody inhibited both omega- and 4-hydroxylation activities in human liver samples that contained relatively high levels of CYP2B6, whereas alpha-naphthoflavone and an anti-CYP1A2 antibody showed inhibitory effects on the 4-hydroxylation activity in a liver microsomal sample in which the CYP1A2 level was relatively high. These results suggest that CYP2B6 has an important role in propofol omega- and 4-hydroxylation in human livers and that the hepatic contents of CYP2B6, CYP3A4, and CYP1A2 determine which P450 enzymes play major roles in propofol oxidation in individual humans.