Sterol 14alpha-demethylase (P45014DM) encoded by CYP51 is a member of the cytochrome P450 (CYP) gene superfamily involved in sterol biosynthesis in fungi, plants, and animals. Constraints imposed by the specific function of CYP51 have severely limited sequence divergence in this family. Consequently, CYP51 is the only P450 family recognizable across all eukaryotic phyla. We have determined the structure of the functional human CYP51 gene, which spans 22 kb, is divided into 10 exons, and maps to 7q21.2-q21.3. The 5' portion of intron 1 is GC-rich and contains potential binding sites for several transcription factors. Primer extension studies reveal predominant transcription initiation sites in liver, kidney, lung, and placenta 250 and 249 bp upstream from the translation start site and a second major site at -100 bp. Ubiquitous expression of human CYP51 (Strömstedt et al., Arch. Biochem. Biophys. 329: 73-81, 1996), the absence of TATA and CAAT patterns, a GC-rich sequence in the promoter region, and initiation of CYP51 transcription at more than one site indicate that CYP51 is a housekeeping gene. The 5'-flanking region, exon 1, and a portion of intron 1 show the characteristics of a CpG island, with the observed/expected CpG ratio of 0.79. Sterol responsive element-like motifs were present in this region, suggesting regulation by oxysterols via a mechanism similar to that associated with other genes involved in cholesterol homeostasis. Comparison of the human CYP51 gene structure with structures of other mammalian and fungal CYP gene families shows that 7 of the 9 CYP51 introns are located at unique positions. More than 80 intron locations exist in mammalian and fungal CYP gene families, and it seems very unlikely that all these introns could have been present in the primordial CYP gene.