This study addresses the hypothesis that transfection of oligonucleotide mimicking a negative regulatory sequence of promoter C of estrogen receptor alpha (ER alpha) gene is sufficient for its re-expression in ER-negative human cancer cell lines. Even if the negative transcription regulator subtracted by the transcription factor decoy is not yet been identified, we demonstrated that after this decoy treatment, the cells produced a functional ER alpha protein able to respond to 17-beta-estradiol and to transactivate a transfected estrogen response element (ERE)-regulated reporter gene. The effects of reactivated ER alpha protein and its estrogen dependence on endogenous target gene expression level, such as ER beta, have been also assessed. The proliferation of the cells transfected with low levels of decoy was significantly increased by estrogen and not by tamoxifen, suggesting that the levels of reactivated ER alpha in these decoy conditions confers a certain hormone sensitivity. On the contrary, high-level expression of ER alpha obtained at high doses of transfected decoy molecule produced a progressive decrease of cell proliferation. Since ER alpha is important in the transcription of different genes and its loss is involved in several pathological processes including neoplastic and chronic diseases, our findings may be of relevance for a possible new therapeutical approach of such diseases.