The promoters of mouse and rat GnRHR genes differ markedly in regard to activin regulation. Activin stimulates the mouse GnRHR promoter, while having no impact on that of the rat. To test whether this difference was due to a single nucleotide change in the rat GRAS homolog we tested a mouse promoter with the rat GRAS homolog and a rat promoter with intact mouse GRAS. The single change in GRAS eliminated activin responsiveness of the mouse GnRHR promoter; however, intact mouse GRAS did not confer activin responsiveness to the rat promoter. Thus, while necessary, GRAS is not sufficient for activin responsiveness of the murine GnRHR promoter. Use of chimeric rat and mouse promoters led to the identification of a 36 bp region residing immediately downstream of GRAS that is necessary for activin responsiveness of the mouse GnRHR gene promoter. Scanning mutagenesis of the 36 bp region localized the functional boundaries of the key regulatory element to adjacent TAAT motifs. The presence of tandem TAAT motifs, the core binding site for multiple members of the homeodomain family of binding proteins, raised the possibility that this region represented a binding site for a homeodomain protein. This region displayed specific binding to a recombinant homeodomain of LHX2. We suggest that GRAS and DARE together define a unique and complex activin/TGF responsive "enhanceosome" whose functional attributes depend on the binding of multiple classes of transcription factors at spatially distinct sites in the promoter of the murine GnRHR gene.