Bone morphogenetic protein 4, BMP4, plays an important role in the development of various organs including the lungs. Little is known regarding the regulation of Bmp4 gene expression in any organ. In the lung, indirect evidence indicates that NKX2.1, a homeodomain transcriptional factor with a demonstrated role in lung morphogenesis, may be a potential upstream regulator of Bmp4 gene expression. In particular, Bmp4 mRNA is reduced or absent in Nkx2.1(-/-) lungs. The human Bmp4 gene has been reported to include two regions of promoter activity in an embryonal carcinoma cell line, Tera2EC. The hBmp4.1 promoter is located upstream of exon I, whereas the second promoter, hBmp4.2, is localized within intron 1 and upstream of exon II. In the current study, we used a co-transfection assay in lung epithelial cells to examine the response of the two hBmp4 promoters to transcriptional stimulation by NKX2.1. Two DNA sequences were identified on the hBmp4.1 promoter that bind NKX2.1 and serve as functional cis-active NKX2.1-responsive elements. Similarly, NKX2.1 stimulated transcription from the hBmp4.2 promoter through two consensus binding sites localized within 412 nucleotides from the site of transcriptional initiation. Thus, both hBmp4 promoters include specific cis-active elements that bind to and mediate transcriptional regulation by NKX2.1. These findings bear functional implications regarding the regulation of a key signaling molecule by a homeodomain transcriptional regulator of lung epithelial morphogenesis.