Gonadotropin releasing hormone (GnRH) is the most upstream regulator of reproduction in vertebrates and its synthesis and release are regulated by gonadal steroid hormones. The proposed sites of hormone action were historically thought to be upstream from GnRH neurons, however, the discovery of ER in a subset of GnRH neurons suggests that this hypothesis should be re-evaluated. To determine a functional role for ER in GnRH neurons, we examined ER's regulation of GnRH promoter activity. The GnRH-producing cell line, GT1-7, was co-transfected with expression vectors containing one of three ER splice variants and a luciferase-reporter construct containing the full-length mouse GnRH promoter sequence or one of two deletions upstream of the transcription start site (-225/-201; -184/-150). Transfected cells were treated with 100 nM 17-estradiol (E₂), diarylpropionitrile (DPN), raloxifene or vehicle. There was a robust increase in GnRH-luciferase activity by all ER splice variants in the absence of hormone. Further, E₂ treatment abolished this response for ER-1 and ER-2, but not ER-13. The -225/-201 and -184/-150 regions were critical for ER-induced promoter activity, as deletion of these regions eliminated the ligand-independent effects of ER. ER-1 binds directly to these promoter regions and because there are no classical estrogen response elements (ERE) in the mouse GnRH promoter, these data raise the possibility that this region contains a novel ERE specific for ER. Overall our data suggest that ER functions as a basic transcription factor in GnRH neurons and demonstrate a potential molecular mechanism for the negative feedback effects of E₂ on GnRH.