Dmrt1is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating theDmrt1proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the ratDmrt1gene and more than 9 kilobases of 5′ flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. TheDmrt15′ flanking region from ?5000 to + 74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for fullDmrt1promoter function. These regions were located between ?3200 and ?2000 base pairs (bp) and downstream of ?150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of ?150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate theDmrt1promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role inDmrt1regulation in the testis.