The estrogen receptors (ERs) bind with high affinity to many structurally diverse ligands by significantly distorting the contours of their ligand-binding pockets. This raises a question: To what degree is ER able to distinguish between structurally related regioisomers and enantiomers? We have explored the structural compliance and specificity of ERα with a set of ligands having a 7-oxa-bicyclo[2.2.1]hept-5-ene sulfonate core and basic side chains typical of selective ER modulators (SERMs). These ligands have two regioisomers, each of which is a racemate of enantiomers. Using orthogonal protecting groups and chiral HPLC, we isolated all 4 isomers and assigned their absolute stereochemistry by X-ray analysis. The 1S,2R,4S isomer has a 80-170-fold higher affinity for ERα than the others, and it profiles as a partial agonist/antagonist in cellular reporter gene assays and in suppressing proliferation of MCF-7 breast cancer cells with subnanomolar potency, far exceeding that of the other isomers. It is the only isomer found bound to ERα by X-ray analysis after crystallization with four-isomer mixtures of closely related analogs. Thus, despite the general compliance of this receptor for binding a large variety of ligand structures, ER demonstrates marked structural specificity and stereospecificity by selecting a single component from a mixture of structurally related isomers to drive ER-regulated cellular activity. Our findings lay the necessary groundwork for seeking unique ER-mediated pharmacological profiles by rational structural perturbations of two different types of side chains in this unprecedented class of ER ligands, which may prove useful in developing more effective endocrine therapies for breast cancer.