DLX3, a member of the large homeobox gene family of transcription factors, is necessary for normal placentation. Targeted deletion of dlx3 in mouse resulted in embryonic death due to placental failure. This study demonstrates the presence of DLX3 mRNA expression in human first trimester and term placental tissue, cultured trophoblast-like cell lines and in isolated primary villous and extravillous trophoblast cells. Using an ovine polyclonal antibody, the spatial distribution was identified for DLX3 in human placental tissues, trophoblast cell lines and in freshly isolated primary trophoblast cells. A 50 kDa immunoreactive DLX3 protein was detected in the human placenta, in trophoblast cell lines and in primary trophoblast cells. Nuclear expression for DLX3 was observed in villous cytotrophoblasts, syncytiotrophoblast and extravillous cytotrophoblast in the proximal regions of the cytotrophoblast cell columns in first trimester placental tissues. Immunoreactivity was also detected in few stromal cells and microvascular endothelial cells surrounding the fetal capillaries. In the first trimester placental bed, DLX3 expression was predominantly observed in the cytoplasm of the endovascular and interstitial trophoblasts. We conclude that the cellular expression of DLX3 was extensive in the human placenta and propose that DLX3 may play an important role in normal placental development.