An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure. A similar structure is present in a synthetic 42 bp oligonucleotide corresponding to the lower strand of a perfect ERE with flanking sequences from the rat prolactin ERE. Moreover, deviations from the ERE consensus sequence decrease the binding of the estrogen receptor to the tertiary-structured ERE. We also have determined that ERE flanking sequences contribute to the affinity of the receptor for the tertiary-structured ERE. Furthermore, ERE flanking sequences can influence the types of interactions that the estrogen receptor makes with the tertiary-structured ERE.