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The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-β (ERβ) and the functional characterization of mouse ERβ protein. ERβ is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-α (ERα) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ERβ and ERα subtypes was demonstrated. Furthermore, ERβ and ERα form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ERβ/ERα heterodimer appears to be dependent on both …