The estrogen receptor (ER) serves as a diagnostic marker for the treatment of breast cancer. Patients with ER-positive breast tumors are likely to respond to hormonal therapies, while ER-negative breast cancers are resistant to endocrine therapies. Most ER-negative tumors do not express detectable levels of ER transcript, highlighting the importance of transcriptional regulation. A novel regulatory element which resembles a steroid hormone response element has been identified in the 5'-flanking region of the human ER gene. We observed 3- to 5-fold higher specific binding to this element in nuclear extracts from ER-expressing MCF-7 breast cancer cells compared to ER-negative MDA-MB-231 breast tumor cells. We termed the factor(s) which bind to this cis-element estrogen receptor upstream binding factor-1 (ERUBF-1). In transient transfection assays in MCF-7 cells, the ERUBF-1 binding site elicited a 20-fold increase in luciferase activity over the ER P1, promoter. This enhancer element was significantly more active in the ER-positive MCF-7 cell line compared to the ER-negative MDA-MB-231 cell line. These data indicate that ERUBF-1 plays an important role in the transcriptional regulation of the ER gene in breast cancer.