Various complementary approaches were used to elucidate the major cytochrome P450 (CYP) enzyme responsible for mifepristone (RU 486) demethylation and hydroxylation in human liver microsomes: chemical and immunoinhibition of specific CYPs; correlation analyses between initial rates of mifepristone metabolism and relative immunodetectable CYP levels and rates of CYP marker substrate metabolism; and evaluation of metabolism by cDNA-expressed CYP3A4. Human liver microsomes catalyzed the demethylation of mifepristone with mean (+/-SD) apparent K(m) and Vmax values of 10.6 +/- 3.8 microM and 4920 +/- 1340 pmol/min/mg protein, respectively; the corresponding values for hydroxylation of the compound were 9.9 +/- 3.5 microM and 610 +/- 260 pmol/min/mg protein. Progesterone and midazolam (CYP3A4 substrates) inhibited metabolite formation by up to 77%. The CYP3A inhibitors gestodene, triacetyloleandomycin, and 17 alpha-ethynylestradiol inhibited mifepristone demethylation and hydroxylation by 70-80%; antibodies to CYP3A4 inhibited these reactions by approximately 82 and 65%, respectively. In a bank of human liver microsomes from 14 donors, rates of mifepristone metabolism correlated significantly with relative immunodetectable CYP3A levels, rates of midazolam 1'-and 4-hydroxylation and rates of erythromycin N-demethylation, marker CYP3A catalytic activities (all r2 > or = 0.85 and P < 0.001). No significant correlations were observed for analyses with relative immunoreactive levels or marker catalytic activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. Recombinant CYP3A4 catalyzed mifepristone demethylation and hydroxylation with apparent K(m) values 7.4 and 4.1 microM, respectively. Collectively, these data clearly support CYP3A4 as the enzyme primarily responsible for mifepristone demethylation and hydroxylation in human liver microsomes.