Recent molecular cloning of estrogen receptor beta (ERbeta) suggests alternative pathways of estrogen signaling, but little is known concerning the role of ERbeta in the development of human breast cancer. In the present study, expression of ERalpha and ERbeta mRNA was determined in a series of chemically transformed human breast epithelial cells as well as various normal and malignant breast cancer cell lines. We observed a very low level of ERbeta expression in the mortal S130 and the spontaneously immortalized MCF10-F human breast epithelial cell lines. As MCF-10F cells were treated with environmental chemical carcinogens, an elevated level of ERbeta expression was observed in the resultant transformed BP1, D3 and BP1-ras cells. An even higher level of ERbeta expression was detected in the more transformed BP1-E, D3-1 and D3-1-ras cell lines. Therefore, results from our study indicate that expression of ERbeta can be induced in chemical carcinogen-transformed human breast epithelial cells, and the more transformed cells showed higher levels of ERbeta expression, regardless of which chemical carcinogens were initially used for cell transformation. These results suggest that expression of ERbeta may contribute to the initiation and progression of chemical carcinogen-induced neoplastic transformation.