Human prostatic steroid 5α-reductase, encoded by theSRD5A2gene on chromosome band 2p23, catalyses the irreversible conversion of testosterone to dihydrotestosterone (DHT), the most active androgen in the prostate, with NADPH as its cofactor. This enzyme has never been purified but a number of competitive inhibitors have been developed for this enzyme since increased steroid 5α-reductase activity may cause benign prostatic hypertrophy and prostate cancer. We report here the detailed biochemical and pharmacogenetic dissection of the human enzyme by analysing 10 missense substitutions and three double mutants which are all naturally found in humans. Nine of these 13 mutants reduce activity (measured asVmax) by 20% or more, three increase steroid 5α-reductase by more than 15% and one results in essentially unaltered kinetic properties suggesting that it is a truly neutral (`polymorphic') amino acid substitution. Substantial pharmacogenetic variation among the mutants was also observed when three competitive inhibitors, finasteride, GG745 (dutasteride) and PNU157706, were investigated. Our studies not only define the substrate and cofactor binding sites of human steroid 5α-reductase, but also have significant consequences for the pharmacological usage of steroid 5α-reductase inhibitors in human patients treated for prostatic conditions.