Estrogen responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor-alpha (ER) and estrogen receptor-beta (ER). Indole-3-carbinol (I3C) strongly down-regulated ER protein and transcript levels, without altering the level of ER protein, in both cell lines. In cells transfected with the ER promoter linked to a luciferase gene reporter, I3C ablated ERpromoter activity. Propyl pyrazole triol (PPT) is a highly selective ERagonist, whereas, 17-estradiol (E) activates both ER and ER. I3C treatment inhibited the PPT and E induced proliferation of breast cancer cells, disrupted the PPT and E stimulation of estrogen response element (ERE) driven reporter plasmid activity as well as of endogeneous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ER, and stimulated the level of ERE binding EReven though the protein levels of this receptor remained constant. In ER-/ER+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a six fold increase in binding of ER to the ERE. I3C also induced ERE and AP-1 driven reporter plasmid activities in the absence of an estrogen receptor agonist suggesting that ER is activated in indole treated cells. Taken together, our results demonstrate that the expression and function of ER and ERcan be uncoupled by I3C with a key cellular consequence being a significantly higher ER:ER ratio that is generally highly associated with anti-proliferative status of human breast cancer cells.