The estrogen receptor, a hormone-regulated transcription factor, regulates gene expression by interacting with a specific nucleotide sequence called the estrogen-responsive element (ERE). In this report we demonstrate by potassium permanganate, osmium tetroxide and diethylpyrocarbonate reactivity and S1 nuclease sensitivity that the nucleotides either within or in the immediate region of imperfect and perfect EREs are in a non-B DNA conformation. The presence of nucleotides in a non-B DNA conformation in the ERE is an intrinsic property of the DNA and is independent of whether the ERE is in linear or supercoiled DNA. S1 nuclease sensitivity was peculiar to the ERE as it was not detected in the thyroid hormone-responsive element. Our results suggest that the nucleotides comprising the ERE are structurally labile. We propose that this intrinsic lability of the ERE could be constrained in vivo such that a unique DNA tertiary structure is formed which may facilitate recognition of the ERE by the estrogen receptor.