The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E 2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5′-GGTCAgagTGACC-3′) but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E 2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E 2-ER binding to AT-rich EREs, not cooperative. The affinity of E 2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E 2-ER binding to closely spaced EREs is more stable ( K d =0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing ( K d =2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E 2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E 2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E 2-ER binding and may be critical for cooperative binding to stereoaligned EREs.