Intracellular levels of active steroid hormones are determined by their relative rates of synthesis and breakdown. In the case of the potent androgen dihydrotestosterone, synthesis from the precursor testosterone is mediated by steroid 5alpha-reductase, whereas breakdown to the inactive androgens 5alpha-androstane-3alpha, 17beta-diol (3alpha-adiol), and androsterone is mediated by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD) and oxidative 17beta-hydroxysteroid dehydrogenases (17beta-HSD), respectively. We report the isolation by expression cloning of a cDNA encoding a 17beta-HSD6 isozyme that oxidizes 3alpha-adiol to androsterone. 17beta-HSD6 is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNAs in mammalian cells is associated with the oxidative conversion of 3alpha-adiol to dihydrotestosterone. Thus, 17beta-HSD6 and RoDH play opposing roles in androgen action; 17beta-HSD6 inactivates 3alpha-adiol by conversion to androsterone and RoDH activates 3alpha-adiol by conversion to dihydrotestosterone. The synthesis of an active steroid hormone by back conversion of an inactive metabolite represents a potentially important mechanism by which the steady state level of a transcriptional effector can be regulated.