The estrogen receptor (ER) is a hormone-regulated transcription factor which is thought to bind to specific DNA sequences as a homodimer. In order to better understand structural requirements for dimerization and its functional role in ER action, we synthesized a series of bivalent ligands based on the non-steroidal estrogen hexestrol. These molecular probes join two hexestrol molecules of the erythro (E, active) configuration with either 4 or 8 carbon linkers (designated E-4-E and E-8-E series, respectively), or with longer linkers comprised of ethylene glycol units (E-eg-E series). Several other bi- and monovalent control compounds were prepared. The bivalent ligands bind to ER with a relative affinity 1-7% that of estradiol. While most of the ligands demonstrated normal monophasic displacement curves in competitive binding assays with [3H]estradiol, uncharacteristic biphasic competitive binding curves were seen for some of the ligands, indicating possible structure-specific, negative site-site interaction. In ER-deficient Chinese hamster ovary (CHO) cells transfected with an expression vector encoding ER, one series of bivalent ligands (E-4-E) had little stimulatory activity and inhibited transcription stimulated by hexestrol, as determined by a transient transfection assay using an estrogen-responsive reporter gene construct [(ERE)2-TATA-CAT, containing two estrogen response elements linked to a TATA promoter and the chloramphenicol acetyl transferase reporter gene]. Monovalent or control bivalent ligands failed to antagonize hexestrol-stimulated activity and were as fully active as hexestrol itself. Studies performed in MCF-7 human breast cancer cells, which contain endogenous ER, yielded similar bioactivity profiles for the E-4-E bivalent inhibitory ligands, showing them to be effective estrogen antagonists, when using either induction of progesterone receptor or (ERE)2-TATA-CAT transcriptional activation as the endpoint. The E-8-E ligand, however, acted as a partial agonist/antagonist of ERE-reporter gene transactivation and a full agonist of progesterone receptor induction in MCF-7 cells, thus showing cell- and response-specific differences in the effects of this bivalent ligand. These bivalent ligands for ER do not show enhanced potency or receptor binding affinity; however, some of them display binding properties that suggest the possibility of structure-specific negative site-site interaction, and some of them function as quite effective estrogen antagonists.