P450c17 is a single cytochrome P450 enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities in the biosynthesis of steroid hormones. We have cloned and sequenced the human P450XVIIA1 gene lying on chromosome 10, which encodes P450c17. The gene spans 6569 bp and is divided into eight exons by seven introns. This intron/exon structure closely resembles that of the P450XXI genes encoding P450c21 (steroid 21-hydroxylase), which contain 10 exons, except that the introns dividing exons 1 and 2 and exons 4 and 5 in the P450XXI gene are absent in the P450XVII gene. Furthermore, computer modeling studies indicate the conformations of P450c17 and P450c21 are very similar. The structures of the P450XXVII and P450XXI genes are very different from other classes of P450 genes. Although the production of P450c17 is under different hormonal, ontogenic, and tissue-specific controls in various types of steroidogenic cells, the adrenal and testis transcribe the P450XVIIA1 gene into P450c17 mRNAs having the same cap sites. S1 nuclease protection experiments locate the principal cap sites as a G residue lying 22 bases 3' to an atypical TTTAAA promoter, and 82 bases 3' to a typical CAAT box. The 5'-flanking DNA contains sequences similar to consensus sequences regulated by cAMP and glucocorticoids.