An important transcriptional regulatory element of the estrogen receptor (ER) gene that binds a protein expressed in ER-positive breast carcinomas has been identified. Using a transient expression assay, we identified a 75-bp region of the 5' untranslated leader of the ER gene which augments expression from the ER promoter. This region contains two binding sites for a protein, estrogen receptor factor 1 (ERF-1), which is expressed in ER-positive breast carcinomas. A concatenated ERF-1 binding site probe identified a 30,000-Da protein. Low-level ERF-1 expression was detected in normal human mammary epithelial cells. Abundant ERF-1 expression was also found in endometrial carcinoma cell lines that express the ER-positive phenotype. These results indicate that ERF-1 expression represents a common mechanism of ER regulation in hormonally responsive carcinomas.