Technical Abstract: Glyceollins (GLYs) are pterocarpan, phytoalexins elicited in high concentrations when soybeans are stressed. We have previously reported that the three most common glyceollin isomers (GLY I, II, and III) exhibit antiestrogenic properties, which may have significant biological effects following human exposure. Of the three isomers, we have also previously shown that the Glyceollin I is the most potent antiestrogen. Glyceollin I was recently synthesized and produced a racemic mixture of two enantiomers, glyceollin I (+/-). Glyceollin I (-) isomer is known to be the enantiomer produced in soybean. In this study, we compared the glyceollin I (+/-) enantiomers ER binding affinity, ability to inhibit estrogen responsive element transcriptional (ERE) activity and endogenous gene expression in MCF-7 cells. In vitro binding studies utilizing recombinant ERa and ERß present both glyceollin I enantiomers with similar relative binding affinity (RBA) for ER subtypes. Reporter gene assays in MCF-7 cells revealed that while (+)-glyceollin I stimulated ERE transcriptional activity, (-)-glyceollin I decreased activity induced by estrogen. Cotransfection reporter assays performed in HEK 293 demonstrated that (+)-glyceollin I increased ERE transcriptional activity of ERa and ERß with and without estrogen with no antiestrogenic activity observed. Conversely, (-)-glyceollin I decreased the activity of both ER subtypes stimulated by estradiol demonstrating potent antiestrogenic properties. Additionally, each Glyc I enantiomer induced unique gene expression profiles in a PCR array panel of genes commonly altered in breast cancer. Taken together, these results demonstrate for the first time the (-)-glyceollin I enantiomer is responsible for the marked antiestrogenic and antagonistic properties observed by glyceollin I.