In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE₂) using human liver microsomes and cytosol. K _m values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 μ M , respectively. V _max/ K _m (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE₂ in human liver. All further studies were performed at a substrate concentration of 0.1 μ M . Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04±1.34, mean±S.D., n =35) and 0.20 to 4.84 (1.20±1.00, n =35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8±4.4, n =21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males ( p <0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 μ M ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6±16.0%, n =27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE₂ and the relative contribution of CYP3A to the 2-hydroxylation of EE₂ in human liver.