Mash-2 expression begins during preimplantation development, but is restricted to trophoblasts after the blastocyst stage. Within the trophoblast lineage, Mash-2 transcripts are first expressed in the ectoplacental cone and chorion, but not in terminally differentiated trophoblast giant cells. After day 8.5 of gestation, Mash-2 expression becomes further restricted to focal sites within the spongiotrophoblast and labyrinth. Downregulation is probably important for normal development since overexpression of Mash-2 reduces giant cell formation. We have investigated the role that the Notch signaling pathway may play in trophoblast development. Mash-2 is a homologue of Drosophila achaete/scute complex genes. In Drosophila, activation of the Notch receptor induces transcriptional repressors encoded by the hairy/Enhancer of split (HES) genes, which interact with the Groucho protein to shut off achaete-scute transcription. In the developing mouse placenta, we found that all elements of the Notch pathway were expressed. In particular, the Notch-2, HES-2, and HES-3 genes were coexpressed in trophoblast giant cells and in foci within the spongiotrophoblast at day 10.5 when Mash-2 transcription becomes restricted. Two members of the mammalian Groucho family were expressed in trophoblasts; TLE3 was expressed broadly in the giant cell, spongiotrophoblast, and labyrinthine regions, whereas TLE2 was limited to giant cells and focal regions of the spongiotrophoblast. These data suggest that Notch signaling through activation of HES transcriptional repressors may play a role in murine placental development.