Cholesterol 7α-hydroxylase (7α-hydroxylase) is a microsomal cytochrome P450 that catalyzes the first step in bile acid synthesis. In this paper, we describe the cloning, characterization, and chromosomal mapping of the human 7α-hydroxylase gene (CYP7). The gene spans 10 kb and contains six exons and five introns. The exon-intron boundaries are completely conserved between the human and rat genes. Sequencing of the 5′ flanking region revealed consensus recognition sequences for a number of liver-specific transcription factors. The human CYP7 gene was mapped to chromosome 8q11–q12 using both mouse-human somatic cell hybrids and in situ chromosomal hybridization studies. A total of four single-stranded conformation-dependent DNA polymorphisms and an Alu sequence-related polymorphism were identified. Of the individuals analyzed, 80% were heterozygous for at least one of these five polymorphisms. The localization and characterization of the human 7α-hydroxylase gene, as well as the identification of polymorphisms, provide the molecular tools necessary to investigate the role of this gene in disorders of cholesterol and bile acid metabolism.