Homeobox genes of the Distal-less (Dlx) family are expressed in the vertebrate embryo in regions where epithelial cell layers contact adjacent mesenchymal cells. This study shows that the human Dlx family member, DLX4, is expressed in the placenta, primarily in regions where epithelial and mesenchymal cell layers contact. In situ hybridization studies at first trimester human placental sections revealed that DLX4 was expressed predominantly in the cytotrophoblast stem cell layer. In term placenta, DLX4 was expressed in the syncytiotrophoblast. Northern analysis revealed two DLX4 transcripts in first trimester placenta of 2.8 and 3.0 kb. Elevated levels of DLX4 mRNA were detected in a choriocarcinoma derived cell line when compared with a cytotrophoblast cell line and normal placenta. This is the first study to show that a member of the Dlx family of homeobox genes is expressed in regions of epithelial and mesenchymal cell layer contact in the human. Accumulated evidence from several studies suggest that a combinatorial code of homeobox genes is required to regulate epithelial-mesenchymal cell interactions in the vertebrate embryo. It is predicted that a similar combination of homeobox genes, that includes DLX4, is involved in regulating epithelial-mesenchymal cell interactions in extraembryonic tissues. DLX4 may also have a role in the regulation of the genes important for trophoblast invasion since the level of expression in trophoblast cell lines reflects invasive potential.