The formation of the trophoblast cell lineage of the placenta is one of the first developmental events to occur in mammalian embryogenesis. To understand the mechanisms of gene regulation in the trophoblast cell lineage we have used the murine adenosine deaminase gene (Ada) as a model. Ada is highly expressed in trophoblast cells of the placenta and is critical for embryo development. A 770bp fragment of the mouse Ada 5' flanking region is capable of directing trophoblast cell-specific expression in a transgenic model system. Earlier studies identified several critical portions of this fragment, including three footprinting regions that are necessary for correct gene expression in the placenta. Using electromobility shift assays (EMSA), we identified a 5bp sequence within footprint 3 that computer databases predicted bound to the transcription factor RUNX1 (also known as acute myeloid leukemia 1). This prediction was confirmed by supershift analysis using antibodies specific for RUNX1. The functional importance of this binding was demonstrated by both transient transfections and transgenic approaches. A significant reduction in expression of the reporter gene in the placenta was seen when the 5bp RUNX1 binding site was mutated. The findings reported here indicate that the RUNX1 transcription factor plays a significant role in regulating Ada gene expression in the trophoblast cell lineage.