Estrogen receptors are derived from two different gene products referred to as estrogen receptor-α (ER-α) and ER-β. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-β binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-α and confer estrogen inducibility to reporter constructs. Binding of ER-α and ER-β occurred at similar DNA concentrations for some EREs, but different DNA concentrations were required to form complexes of the two receptors with other elements. These results illustrate for the first time by direct receptor-DNA binding studies that both ER-α and ER-β bind to a number of EREs present in endogenous hormone regulated genes, and further suggest that the two forms of the receptor display different patterns of affinities for naturally occurring hormone response elements.