The trophectoderm epithelium of the blastocyst is the first tissue to differentiate in the mammalian embryo and gives rise to all cells of the trophoblast lineage. These cells form the majority of the fetal component of the placenta, which is critical for intrauterine development. Our interest in the molecular determinants of trophoblast lineage development has led us to undertake an analysis of the regulatory region of a trophoblast-specific gene termed 4311. The transcription start site was mapped by both primer extension and RNase protection analyses. We show that cloned 4311 upstream sequences are able to confer the correct temporal and spatial expression pattern of 4311 on a reporter gene in transgenic mice. In addition, we have characterized a 340-bp region, 3.7 kb upstream of the transcription start, that is sufficient for the specific expression of lacZ in cells of the developing spongiotrophoblast in transgenic mice. Delineation of a diploid trophoblast-specific regulatory region will facilitate the identification of factors that may regulate the development of this lineage.