Estrogen receptors alpha and beta (ER and ER) mediate the actions of estrogens in a variety of normal and cancer target cells. Estrogens differ in their preference for these ERs, and many phytoestrogens bind preferentially to ER. To investigate how phytoestrogens such as genistein impact ER-regulated gene expression, we used adenoviral gene delivery of ER coupled with ER–depletion with siRNA to generate human breast cancer (MCF-7) cells expressing four complements of ER and ER. We examined the dose-dependent effects of genistein on genome-wide gene expression by DNA microarrays and monitored the recruitment of ERs and coregulators to responsive regions of estrogen-regulated genes. At a low (6 nM) concentration, genistein regulated gene expression much more effectively in cells coexpressing ER and ER than in cells expressing ER alone, whereas at high concentration (300 nM) genistein induced transcriptome changes very similar to that of 17-estradiol. We demonstrate that ER is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites, and that differential occupancy of ER and ER by genistein and E2 in turn influences the recruitment patterns of coregulators such as SRC3 and RIP140. Our observations indicate that genistein is a potency-selective ligand for gene expression regulation by ER and ER, and that the ability of ER and ER to serve as determinants of gene expression is greatly influenced by the nature of the ligand, by ligand dose, and by the differential abilities of ligand-ER complexes to recruit different coregulators at ER binding sites of hormone-regulated genes.