Sterol 12α-hydroxylase (CYP8B1) is a hepatic cytochrome P-450 that controls the ratio of cholic acid over chenodeoxycholic acid in bile and thus controls the solubility of cholesterol. Both the human and the mouse CYP8B1 complementary DNA and gene were cloned and structurally characterized. Surprisingly, the genomic DNA from both species was found to lack introns. The major transcript of the human gene was estimated to be 3950 bp, and the putative promoter region was estimated to be at least 1360 bp. The murine structural gene was found to span approximately 3 kb. By using FISH and radiation hybrid mapping techniques, the humanCYP8B1gene was located to chromosome 3p21.3–p22, whereas FISH mapped the murine counterpart to chromosome 9qF4, a region that is homologous to the third human chromosome. The results from the chromosome mapping and Southern blotting indicated that the gene is present in a single copy. Transcription of the mouse and humanCYP8B1genes was initiated from a position situated 51 and 35 bases, respectively, downstream of a consensus TATA box. A homology of 21% for the promoter regions of mouse and human may indicate differences in transcriptional regulation. Although a potent induction of CYP8B1 mRNA was observed upon starvation of mice, the mechanism behind this effect was not revealed by analysis of the promoter for potentialcis-acting elements. In the human promoter, several possiblecis-acting regions were identified but none of them could be directly related to bile acid metabolism. After transfection of COS cells with the human coding region, mRNA and enzymatic activity for the 12α-hydroxylase were identified. This is the first mammalian cytochrome P-450 gene reported to lack introns. The importance of this structural feature for evolution and gene regulation is discussed.