Estrogen (17β-estradiol, E2) plays pivotal roles in the function and maintenance of the skeleton, including the bone-forming osteoblasts (OBs). The functions of E2 are largely mediated through two distinct estrogen receptor isoforms, ERα and ERβ, both of which are expressed in OBs. The level of each isoform dominates at early or late stages of OB differentiation. To date, only a limited comparison between the transcriptional targets of ERα and ERβ on endogenous gene expression has been reported. We have developed new stable cell lines, which contain doxycycline (Dox)-inducible ERα and ERβ, in the U2OS human osteosarcoma to determine the global transcriptional profile of ERα- and ERβ-regulation of endogenous gene expression. The U2OS-ERα and U2OS-ERβ cell lines were treated with Dox and either vehicle control or E2 for 24 h. Gene expression analysis was performed using a microarray containing ∼6,800 full-length genes. We detected 63 genes that were regulated solely by ERα and 59 genes that were only regulated solely by ERβ. Of the ERα-regulated genes, 81% were upregulated and 19% were inhibited. Similarly 76% of the ERβ-regulated genes were upregulated whereas 24% were inhibited by E2. Surprisingly, only 17 genes were induced by both ERα and ERβ. Real-time PCR was employed to confirm the expression of a selected number of genes. The regulation of a number of known E2-responsive genes in human OBs, as well as many interesting novel genes, is shown. The distinct patterns of E2-dependent gene regulation in the U2OS cells by ERα and ERβ shown here suggest that during OB differentiation, when either isoform dominates, a unique pattern of gene responses will occur, partially due to the differentiation state and the ER isoform present. J. Cell. Biochem. 90: 315–326, 2003. © 2003 Wiley-Liss, Inc.