Dominant negative estrogen receptors are transcriptionally inactive, altered forms of the estrogen receptor (ER) that can dimerize with the ER and have the potential to inactivate the biological functions of this receptor. Here, we provide the first report that adenoviral delivery of a dominant negative ER to ER-positive breast cancer cells is able to effectively suppress estrogen-stimulated cell proliferation and the hormonal induction of endogenous genes. We constructed recombinant adenoviral vectors expressing a dominant negative ER (S554 fs, Ad-fs) or, for comparison, antisense ER (Ad-AS), or the sense wild-type ER (Ad-WT). Expression of the dominant negative ER or antisense ER, but not wild-type ER, blocked estradiol stimulation of the estrogen-responsive genes pS2 and c-myc. The dominant negative ER also fully abolished the estradiol-induced increase in proliferation of MCF-7 breast cancer cells, as did the antisense ER. The antiproliferative effects of the dominant negative and antisense ERs are explained by an increase in the number of cells in the G0/G1 stage of the cell cycle and decrease in the number of cells in G2/M as determined by flow cytometry, and also by a significant increase in the percentage of cells undergoing apoptosis. Our data strongly support the idea that targeting ER action using recombinant viral delivery of dominant negative ERs is an effective way to suppress ER-positive breast cancer cell proliferation and suggests the potential attractiveness of dominant negative gene therapy approaches targeted to the ER for the treatment of hormone-responsive breast cancer.