Nkx2.1 encodes a homeodomain transcription factor whose expression is restricted to the thyroid, lung and specific regions of the forebrain. NKX2.1 plays a key role in the development of the latter organs. In lung epithelial cells, two regions of promoter activity, designated as proximal and distal promoters, map to DNA elements located upstream of exons 1 and 2 (within intron 1). That both promoters are active in vivo has been demonstrated by the presence of multiple Nkx2.1 mRNA species with distinct and appropriate exonic composition. The mechanisms of Nkx2.1 tissue selective gene expression remain entirely unknown. We have examined the potential of three overlapping DNA fragments, representing a total of approximately 4 kb of potential regulatory DNA from the baboon Nkx2.1 5′ flanking region to direct expression of LacZ in transgenic mice during embryonic development. The three constructs include sequences in proximal, distal and both promoters separately. All three fragments directed LacZ expression to the brain of transgenic E15 and E18 mouse embryos. In addition to a number of other sites, all three constructs were active in subgroups of cells localized in the hypothalamus, a well-established site of endogenous Nkx2.1 gene expression. Two of the fragments conferred tracheal epithelial-specific LacZ gene expression, but parenchymal lung expression was not observed. None of the three fragments had activity in the thyroid. These data demonstrate the complexity of the Nkx2.1 tissue specific gene regulation and suggest that cis-active elements required for tracheal versus lung morphogenesis may be distinct. The same applies to the brain, which provides the most permissive environment for recognition of Nkx2.1 tissue specific cis-active elements.