Trophoblast cells arising from the implanted blastocyst form the fetal component of the maternal-fetal interface throughout pregnancy. Previous in situ hybridization studies have shown that some subpopulations of these cells, cytotrophoblast cells within and exterior to placental villi, contain class I HLA mRNA. Those studies, performed under moderate conditions of stringency, did not determine which member(s) of the class I HLA gene family was transcribed. In this study, in situ hybridization experiments were conducted under conditions of high stringency using biotinylated antisense and sense RNA probes specific for HLA-G and HLA-E. HLA-G mRNA was identified in first trimester cytotrophoblast cells and in term chorionic membrane cytotrophoblast cells, but was low to undetectable in syncytiotrophoblast of both early and late gestation placentas. Placental villous mesenchymal cells in first trimester but not term placentas contained HLA-G transcripts. HLA-E mRNA was clearly identified only in small round cells present in first trimester decidua and term membranes. These experiments provide the first direct evidence for transcription of the HLA-G gene by cytotrophoblast cells in situ. Expression of this nonpolymorphic gene in place of HLA-A,B,C by trophoblast cells exposed to maternal blood and tissues may allow the juxtaposition of genetically disparate cells required for human pregnancy.