The level of estrogen receptor (ER) is a key determinant for the management of ER-positive [ER(+)] breast cancer patients. Growth of many human breast cancers is regulated by estrogen (E2) and progesterone (Pr). Generally, the ER in ER(+) breast cancer patients is targeted for therapy with antihormones. However 40% of ER(+) patients do not respond to antihormone therapy. Thus, the identification of antihormone resistant ER(+) breast cancers is essential for therapeutic predictions. Although 3H-E2 binding and immunodetection can identify ER, these procedures do not assess the functional state of the receptor molecule. In this study we describe a novel and rapid assay for the detection of ER and its functional state on the basis of the downstream interaction with its response element (ERE) based on the preferential binding of DNA-protein complex (ERE-ER) to a nitrocellulose membrane (NMBA). This method permits measurement of both the total and the functional fraction of ER. The ER status was examined in breast cancer cell lines and in breast cancer biopsy specimens by (i) 3H-E2 binding assay, (ii) immunodetection assays and (iii) by its interaction with 32P-ERE. The sensitive NMBA assay was validated with well-characterized ER(+) breast cancer cell lines and also identified functional variants of ER among breast tumor biopsy specimens.