Mestranol, the estrogen component of some oral contraceptive formulations, must be demethylated to its active metabolite, 17 alpha-ethinyl estradiol, to produce estrogenic activity. To investigate the transformation of mestranol to ethinyl estradiol, an in vitro assay was used with human liver microsomes from four different donors. Incubation of a fixed concentration of mestranol (3 mumol/L) with varying concentrations of CYP inhibitors revealed strong inhibition of ethinyl estradiol formation by sulfaphenazole, a specific CYP2C9 inhibitor, with an average inhibitor concentration at one half of Emax (IC50) of 3.6 mumol/L (range, 1.8-8.3 mumol/L) and an average maximal inhibitory capacity (Emax) of 75% (range, 60-91%). Troleandomycin (a CYP3A3/4 inhibitor) and quinidine (a CYP2D6 inhibitor), however, produced no substantial inhibitory activity. alpha-Naphthoflavone (a CYP1A1/2 inhibitor only at concentrations < 2 mumol/L and a CYP2C9 inhibitor at higher concentrations) had a weak inhibitory effect on ethinyl estradiol formation (< 20% decrease in mestranol demethylation activity). Of the three antifungal azoles tested, miconazole strongly inhibited mestranol demethylation, with an average IC50 of 1.5 mumol/L (range, 0.7-3.2 mumol/L) and an average Emax of 90% (range, 77-100%), whereas fluconazole displayed relatively weak inhibition only at the highest concentration of 50 mumol/L (mean reduction in demethylation activity was 29%). Itraconazole produced no meaningful inhibition. Strong inhibition of ethinyl estradiol formation by sulfaphenazole suggests a major contribution of CYP2C9 to this reaction.