Estrogen signaling is mediated by ER alpha and -beta. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ER alpha and ER beta bind to an ERE independently from ER ligands. We found that although the binding affinity of ER beta for an ERE is 2-fold lower than that of ER alpha, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ER alpha preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ER alpha is more transcriptionally active than ER beta. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ER alpha to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.