The best known activity of steroid 5 alpha-reductase is the transformation of testosterone into dihydrotestosterone, the most potent androgen. Two types of human steroid 5 alpha-reductase cDNAs and the type I gene have previously been isolated and characterized. This report describes the isolation and characterization of the human type II 5 alpha-reductase gene, the gene most likely responsible for male pseudohermaphroditism due to 5 alpha-reductase deficiency as well as the one presumed to be involved in a major androgen-related diseases such as prostate cancer and benign prostatic hyperplasia. The type II 5 alpha-reductase gene contains five exons of 352, 164, 102, 151 and 1695 bp, respectively, which share 43.8% to 64.1% homology with exons of the corresponding type I gene. These exons are separated by four introns of greater than 29, and approximately 2.3, 2.0 and 3.0 kb. Analysis of primer extension products by polyacrylamide gel electrophoresis as well as by subcloning and sequencing reveals a start site located 71 nucleotides upstream the ATG initiating codon.