The transcription factor Elf3, which is one of over 25 Ets family members, is expressed in a wide variety of carcinomas and has been shown to promote the transcription of many genes implicated in cancer. To understand how the Elf3 gene is regulated at the transcriptional level, we probed its 5′-flanking region, and we report here the identification of both proximal and distal regions that regulate murine Elf3 promoter activity. In addition to mapping the transcription start site of the Elf3 gene, the work described in this study identifies four cis-regulatory elements in the proximal promoter region of the gene. These include a cis-regulatory element previously designated ESE, a κB site, a POU motif, and a CCAAT box. In addition, we demonstrate that a novel 94 bp region 2 kb upstream of the transcription start site significantly elevates Elf3 promoter activity in F9-differentiated cells, but not in the parental F9 embryonal carcinoma (EC) cells. This region appears to be largely responsible for the increase in Elf3 promoter activity that accompanies the differentiation of embryonal carcinoma cells.