Estrogen receptors alpha and beta (ERalpha and ERbeta) 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 ERbeta. To investigate how phytoestrogens such as genistein impact ER-regulated gene expression, we used adenoviral gene delivery of ERbeta coupled with ERalpha depletion with small interfering RNA to generate human breast cancer (MCF-7) cells expressing four complements of ERalpha and ERbeta. 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 ERalpha and ERbeta than in cells expressing ERalpha alone, whereas at high concentration (300 nm), genistein induced transcriptome changes very similar to that of 17beta-estradiol. We demonstrate that ERbeta is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites and that differential occupancy of ERalpha and ERbeta by genistein and 17beta-estradiol in turn influences the recruitment patterns of coregulators such as steroid receptor coactivator 3 (SRC3) and receptor-interacting protein 140 (RIP140). Our observations indicate that genistein is a potency-selective ligand for gene expression regulation by ERalpha and ERbeta and that the ability of ERalpha and ERbeta 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.