Cholesterol 7α-hydroxylase is the rate limiting enzyme in bile acid biosynthesis and plays an important role in cholesterol homeostasis. The Golden Syrian hamster has been used as an animal model for the study of atherosclerosis and cholesterol gallstone disease. We have screened a λ DASH II hamster liver genomic library using a rat cDNA as a hybridization probe. A 14-kb genomic clone has been isolated and characterized by restriction mapping and Southern blot hybridization. The clone contained the full-length gene encoding cholesterol 7α-hydroxylase together with an upstream sequence of approximately 5 kb. DNA sequencing and analysis of about 11 kb of the gene revealed that the hamster CYP7 gene consists of six exons and five introns, which have the same structures and sizes as predicted in the rat and human CYP7 genes. The nucleotide and deduced amino acid sequences of the hamster cholesterol 7α-hydroxylase have a high sequence identity of about 90% to the rat and 82% to the human sequences. Particularly, exons 2, 5, and 6 are highly conserved among these species, thus reflecting the presence of some domains that are crucial for the activity of this unique enzyme. The putative cholesterol-binding region, an aromatic amino acid region, and the P450 heme-binding region are completely conserved. Comparison of the 250-bp 5′-flanking sequence to the corresponding region in the rat and human genes revealed a high degree of homology ranging between 71% and 82%. Next to the canonical TATA and CCAAT boxes are many consensus sequences (LF-A1, LF-B1, TGT3) for liver-specific or -enriched transcription factors (HNF4, HNF1, and HNF5, respectively) and an imperfect direct repeat of thyroid hormone responsive element (TRE), which is located between TGT3 and LF-B1. These sequence motifs are completely conserved among the rat, human, and hamster CYP7 genes. Several modified sterol regulatory element (SRE)-like sequences are located in the upstream flanking region and in the first intron. This highly conserved proximal promoter may play important roles in the transcription activity and in the regulation of the CYP7 gene by physiological agents, such as bile acids and steroid/thyroid hormones. This is the first report describing the complete nucleotide sequence and confirming the structure of a CYP7 gene. This detailed structural information and analysis of the gene sequence will allow us to study the transcriptional regulation of such an important gene for cholesterol homeostasis.