Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.