Proliferation of breast cancer cells is mediated by estrogen receptors (ER)-ERalpha and ERbeta. At present, contradictory observations complicate the understanding of involvement of ERbeta in breast cancer and functional definition of ERbeta as a prognostic marker. A stable expression of full length ERbeta was established in the ERalpha-positive MCF-7 breast carcinoma cell line to evaluate the role for ERbeta in maintenance of cell viability and estrogenic response, as well as proliferation, morphology and cell cycle progression. In order to verify in vivo tumourigenicity of ERbeta transfectants were transplanted into nude mice. Transfection of ERbeta in MCF-7 resulted in a marginal increase of gelsolin protein expression. Constitutive expression of ERbeta resulted in a significant 30% inhibition of cellular growth compared with transfection of the mock vector alone (p=0.043). This reduction in growth was associated a retardation of transition into S-phase of the cell cycle. The in vitro response to 17beta-estradiol was reversed in cells over-expressing ERbeta (p=0.016). However, no difference in response to the antiestrogens tamoxifen and ICI 182,780 was observed in the presence of ERbeta. Importantly, over-expression of ERbeta prevented establishment and growth of tumours as subcutaneous xenografts in immunodeficient mice in vivo. These observations support the notion that ERbeta is a tumour suppressor and is exploitable in terms of cancer prevention, improving therapeutic response or predicting disease progression.