Sterol-regulatory element binding protein (SREBP) 1 and SREBP2 are ubiquitously expressed transcription factors that play key roles in the regulation of cholesterol and fatty acid metabolism. SREBP1 and SREBP2 share approximately 47% sequence identity and map to chromosomes 17 and 22, respectively. The gene encoding SREBP1 (SREBF1) has been cloned and characterized. In this paper we describe the gene structure and 5'-flanking sequence of SREBF2. SREBF2 spans 72 kb and is composed of 19 exons and 18 introns. The locations of the exon/intron boundaries of SREBF2 are remarkably similar to those of SREBF1, but SREBF2 is approximately 2.8 times larger in size. The 5'-flanking regions of SREBF2 and of two alternatively spliced forms of SREBF1, SREBF1a and SREBF1c, were sequenced, and the SREBF2 transcription start site was determined. A perfect 10-bp sterol regulatory element (SRE)-1 sequence was present in the promoter region of SREBF2. No SRE-1 was identified in the 5'-flanking sequences of either SREBF1a or SREBF1c, but several E-box sequences were present in SREBP1c. Thus, analysis of the 5'-flanking regions provides support that these two transcription factors, though similar in their coding sequence and overall gene structure, have different physiological roles. Finally, evidence is presented for the presence of another expressed gene of unknown function located 500 bp upstream of SREBF2.