[h=2]Abstract[/h] The major products of testosterone, androstenedione, and progesterone metabolism by human epidermal keratinocytes are 5α-reduced steroids, viz. 5α-dihydrotestosterone, 5α-androstanedione, and 5α-dihydroprogesterone, respectively. The rates of metabolite formation by these cells were linear with incubation time up to 3 h. The apparent K[SUB]m[/SUB] of keratinocyte 5α-reductase was 1.3 ÂµM for androstenedione and 1.5 μM for progesterone. 5α-Reductase activity was found only in particulate subcellular fractions of a homogenate of epidermal keratinocytes when assayed with tritium-labeled progesterone as the substrate and NADPH as the cofactor. In addition to 5α-reductase activity, other enzymatic activities found in epidermal keratinocytes were 17β-hydroxysteroid oxidoreductase and 3β-hydroxysteroid oxidoreductase. These enzymes were expressed in the formation of androstenedione from testosterone, testosterone from androstenedione, isoandrosterone from androstenedione, and 3β-hydroxy- 5α-pregnan-20-one from progesterone. The apparent K[SUB]m[/SUB] of 17β-hydroxysteroid oxidoreductase for androstenedione in epidermal keratinocytes was 10μM. When measured at weekly intervals, the rates of product formation from testosterone, androstenedione, or progesterone by cultured epidermal keratinocytes increased several-fold with advancing time in culture up to 3 weeks. The results of these studies suggest that epidermal keratinocytes are a major site of synthesis of biologically potent androgens in human skin, viz. testosterone from androstenedione and 5α-dihydrotestosterone from testosterone. Skin is a target organ for 5α-dihydrotestosterone action, and thus, thelocal formation of 5α-dihydrotestosterone may play an important role in the regulation of proliferation and differentiation of keratinocytes.