Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors α and β (ERα and ERβ). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERα or ERβ, at levels comparable with those in osteoblasts, and we characterized their response to 17β-estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the approximately 100 regulated genes we identified, some were stimulated by E2 equally through ERα and ERβ, whereas others were selectively stimulated via ERα or ERβ. The E2-regulated genes showed three distinct temporal patterns of expression over the 48-h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERβ, in keeping with the selective E2 enhancement of the motility of ERβ-containing cells. On genes regulated equally by E2 via ERα or ERβ, the phytoestrogen genistein preferentially stimulated gene expression via ERβ. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.