Technical Abstract: Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be a novel antiestrogen which bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogen-stimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II and III) we have identified the active antiestrogenic component using competition binding assays with human ERa and in an estrogen responsive element-based luciferase reporter assay (ERE-Luc). We identified glyceollin I as the active component of the combined glyceollin mixture. Molecular modeling of glyceollin I, II and III docking to the ERa ligand binding cavity demonstrates a unique Type II antiestrogenic confirmation adopted by glyceollin I, but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen (4-OH-Tam) and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17ß-estradiol (E2) stimulated expression of progesterone receptor (PgR) and stromal derived factor-1a (SDF-1). Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent of hormone-dependent tumors.