Biochanin A and formononetin are abundant in legumes. These proestrogenic isoflavones can be converted by 4'-O-demethylation to the more potent phytoestrogens genistein and daidzein. Incubation of biochanin A or formononetin with human liver microsomes resulted in 4'-O-demethylation and the production of additional metabolites. Three new hydroxylated formononetin derivatives, 6,7-dihydroxy-4'-methoxyisoflavone, 7,8-dihydroxy-4'-methoxyisoflavone, and 7,3'-dihydroxy-4'-methoxyisoflavone, were isolated and characterized. We surveyed the O-demethylase competence of cytochrome P450 isoforms found in human liver. Human cytochrome P450 isoforms 1A2, 2E1, 2C9*1, 2C19, and 2D6*1 catalyzed biochanin A consumption and genistein production. Human cytochrome P450 isoforms 1A2, 2C9*1, 2A6, 2D6*1, and 2C19 catalyzed formononetin consumption and daidzein production. These isoforms also generated other hydroxylated metabolites. Although O-demethylation of isoflavones has been attributed to metabolism by gut microflora, our study demonstrates that human hepatic microsomal enzymes can perform the same transformation and may play a key role in the conversion of 4'-O-methylated isoflavones to more potent phytoestrogens.