DMRT1 is a transcription factor expressed only in Sertoli cells and undifferentiated spermatogonia of the postnatal testis, where it is required for proper cellular differentiation and fertility. To elucidate the transcriptional regulatory regions that provide DMRT1's cell-specific expression, transgenic mice containing a LacZ reporter gene driven by variable amounts of rat Dmrt1 5′ flanking sequence, 9 kb and smaller, were evaluated. Examination of transgene expression by RT-PCR indicated that multiple promoter regions direct Dmrt1 to the testis and that sequences upstream of 2.8 kb are needed for both Sertoli cell expression and limiting transcriptional influence imposed by surrounding chromatin. Thus, whereas many of the transgenes were expressed in the testis, the ones with smaller promoters were significantly more prone to expression at ectopic sites or to complete silencing. Transgene expression in Sertoli cells and germ cells was assessed by immunohistochemistry and RT-PCR following busulfan treatment to remove germ cells. Both evaluations indicated expression of the 9- and 3.2-kb promoters in Sertoli cells and germ cells, whereas activity of smaller promoters was largely restricted to germ cells. In all, the present study provides in vivo evidence that distinct promoter sequences participate in Dmrt1 regulation in somatic cells and germ cells, with the −3.2 kb/−2.8 kb region directing expression in Sertoli cells and downstream sequences (≤1.3 kb) directing it in germ cells. Further exploration of the mechanisms restricting Dmrt1 expression to the testis revealed that FOXL2, a transcription factor required for differentiation of the ovary, repressed Dmrt1 promoter through the −3.2 kb/−2.8 kb regulatory region, offering a potential mechanism for Dmrt1 transcriptional silencing in granulosa cells.