Consistent with the common role of Nkx6 family members in specifying motor neuron identity, we show that over-expression of Drosophila Nkx6 results in an increase in the number of Fasiclin II expressing motor neurons in the intersegmental nerve B branch. Our dissection of the regulatory domains of Nkx6 using chimeric cell culture assays revealed the presence of two repression domains and a single activation domain within this transcription factor. As well as its conserved homeodomain, Nkx6 also has a candidate Engrailed homology 1 (Eh1) domain that is conserved amongst all NKx6 family members, through which vertebrate NKx6-type proteins bind the co-repressor, Groucho (Muhr, J., et al., 2001. Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104, 861-73). Paralleling our previous reports that the Eh1 domain of Vnd and Ind are ineffective in Gal4 chimeric assays (Von Ohlen, T., Syu, L.J., Mellerick, D.M., 2007. Conserved properties of the Drosophila homeodomain protein. Ind. Mech. Dev. 124, 925-934; Yu, Z., et al., 2005. Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator. Nucleic Acids Res. 33, 1-12), we found that the Eh1 domain of Nkx6 did not significantly enhance repression in Gal4 chimeric assays. However, when we performed co-immunoprecipitation analyses, we found that Nkx6 can bind Groucho and that binding of Nkx6 to this co-repressor is modulated intra-molecularly. Full length Nkx6 interacted with Groucho poorly, because sequences at the carboxyl terminal of NKx6 interfere with Groucho binding, despite the presence of the Eh1 domain. In contrast, a carboxyl terminal Nkx6 deletion bound Groucho strongly. In keeping with the presence of an activation domain within Nkx6, we also report that Nkx6 can activate reporter expression driven by an Nkx6.1 enhancer that mediates auto-activation in transient transfection assays. The presence of multiple repression domains in Nkx6 supports Nkx6's role as a repressor, potentially using both Groucho-dependent and independent mechanisms. Thus, Nkx6 likely functions as a dual regulator in embryos.