Hormone-independent human breast cancer is characterized by estrogen receptor (ER) loss and the acquisition of high epidermal growth factor receptor (EGFR) levels. Despite the tendency for an inverse correlation between EGFR and ER, EGFR is a strong prognostic indicator for poor survival rate independent of ER status suggesting that EGFR overexpression is an important step in the progression to estrogen independence. We have previously shown that several DNase I hypersensitive sites which correspond to potential regulatory regions reside within the EGFR gene first intron exclusively in hormone-independent breast cancer cells. CAT assays investigating the transcriptional activity of the first intron of EGFR indicate that a 140 bp region has an enhancer ability specifically in these hormone-independent breast cancer cells. The DNA-protein interaction that occurs in this enhancer was localized to a 35 bp region and displayed enhancer-like activity in the same hormone-independent breast cancer cells. Furthermore, the protein that binds to this 35 bp region seems to be ubiquitous in the cell lines tested but in higher abundance in high EGFR expressing cells. Identifying the specific regulatory elements involved in EGFR up-regulation could lead to the development of therapies for preventing and treating estrogen-independent breast cancer.