We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.