Currently, the therapy for breast cancer is determined by immunohistochemical staining of the primary tumour for oestrogen receptor alpha (ERα). However, a proportion of ERα-positive patients fail to respond to tamoxifen and a proportion of ERα-negative patients show response. Here, we describe a novel procedure for the purification of malignant breast epithelial cells in an attempt to identify these patients at an early stage. Using this procedure, we are able to purify malignant cells to >90% purity as determined by immunohistochemical staining, cytology and fluorescent in situ hybridisation (FISH). While the malignant cells can be maintained in culture they do not proliferate in contrast to purified breast epithelial cells from reduction mammoplasties. Moreover, ERα and progesterone receptor (PR) expression is maintained in malignant cells, whereas normal epithelial cells rapidly lose ERα and PR. Functional studies were performed on the separated malignant cells in terms of their response to oestradiol and tamoxifen. Four out of the seven ERα-positive tumours showed a significant reduction in cell numbers after tamoxifen treatment compared to oestradiol, ERα negative tumours failed to show a response. We conclude that (a) it is possible to purify and maintain breast cancer cells for a sufficient period to permit functional studies and (b) ERα is retained in culture facilitating the use of these cells in studies of the mechanism of endocrine response and resistance in vitro.