Estrogen receptors activate transcription in part through direct interactions with specific DNA motifs, called estrogen response elements (EREs). Here we show that the strong and sustained induction of the gene regulated in breast cancer 1 (GREB1), a gene of unknown function that has been previously suggested to play a role in the effects of estradiol on breast cancer cell proliferation (Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., and Lippman, M. E. (2005) Breast Cancer Res. Treat 92, 141-149), is mediated by binding of estrogen receptor alpha (ERalpha) to three consensus EREs spread over approximately 20 kb of upstream flanking sequences. In addition to ERalpha, coactivator SRC-3, acetylated histones and phosphorylated RNA polymerase II (P-polII) were detected on all three EREs in the presence of estrogen, while basal recruitment of ERalpha and P-polII was observed only on the proximal element. Chromatin loops were formed between each ERE and the GREB1 transcriptional start site in the presence of estrogen but not of a total antiestrogen. Furthermore, estradiol induced physical association between EREs, suggesting that these elements function as a potent multipartite enhancer to regulate GREB1 transcription.