The metabolic activation of estrone (E1), a potent estrogen was investigated using recombinant human cytochrome P450 enzymes, 1A2, 2B6, 2C8, 2C9, 2C9R144C, 2E1, 3A4, 3A5 and liver microsomes from 14 human organ donors. At least five products of E1 were detected and quantitated by HPLC and gas chromatography-mass spectrometry (GC-MS). Among these metabolites, 16alpha-OH-E1, 2-OH-E1 and 4-OH-E1, which are believed to be associated with estrogen carcinogenesis in animals, were definitively identified. Of all P450s examined, 1A2 and 3A4 exhibited the highest activities with turnovers of 3.4 and 2.5 nmol/min/nmol P450 for the total metabolism of E1, respectively, while 3A5, 2C9 and 2C9R144C showed moderate activities. 2B6, 2E1 and 2C8 did not produce any significant amount of products. 1A2 formed almost exclusively the 2-OH-E1 at a rate of 3.3 nmol/min/nmol but 3A4 preferentially formed the metabolite X1 (an unknown hydroxylation product) and 16alpha-OH-E1. Kinetic characterization showed that the Km values of 1A2, 3A4 and 3A5 were 14, 95 and 64 microM and Vmax were 5.43, 0.68 and 0.35 min(-1), respectively. All human liver microsomes were capable of metabolizing estrone and a 4-fold variation was seen between individuals. The relative amount of metabolites formed was generally 2-OH-E1 > metabolite X1 > 4-OH-E1 > 16alpha-OH-E1 > metabolite X2. 3A4/5 enzyme complex was assessed by inhibitory monoclonal antibody specific for 3A4/5 to contribute 60-88% to the formation of individual metabolites in human liver except for 2-OH-E1 (3%). The formation of 2-OH-E1 and 16alpha-OH-E1 by 14 human liver microsomes was significantly correlated with caffeine 3-demethylation supported by 1A2 (r2 = 0.87) and with testosterone 6beta-hydroxylation by 3A4 (r2 = 0.66), respectively. Thus the metabolic patterns exhibited by human liver are likely due to the combined activities of the P450 1A2 and 3A4 enzymes.