Homeobox genes are a large family of transcription factors. Of these, the HLX homeobox gene (previously known as HLX1 and HB24) is important for normal placentation. We have previously shown that HLX mRNA expression is significantly reduced in fetal growth-restricted human placentae compared with control placentae. In this study, a rabbit polyclonal antibody to the homeodomain protein HLX was raised and characterised. Western analysis revealed a protein of 50 kDa. HLX protein was detected in cellular nuclei in the cytotrophoblast-derived cell lines HTR8/SVneo, SGHPL-4, JEG-3, JAR and BeWo. Dual labelling with cytokeratin 7 was used to determine the spatial distribution of HLX in the early placenta and fetal membranes, showing both a perinuclear and punctate nuclear distribution for HLX. In the early pregnancy placenta HLX was localised to villous cytotrophoblast, and extravillous cytotrophoblast nuclei in the proximal regions of the cytotrophoblast cell columns, but was not detected at significant levels in the syncytiotrophoblast. In first trimester placental bed biopsies, HLX expression was not localised to the nucleus but instead was found in the cytoplasm. We conclude that HLX is primarily expressed in cytotrophoblast cell types in the human placenta and propose that HLX is involved in cytotrophoblast proliferation and downregulation of cell differentiation.