Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto- and 20-ketosteroid reductases or as 3-, 17- and 20-hydroxysteroid oxidases. These AKRs can either convert potent sex hormones (androgens, estrogens and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the pre-receptor level. AKR1C2 is expressed in prostate and in vitro it will catalyze the NAD⁺-dependent oxidation of 3-androstanediol (3-diol) to 5-DHT. This reaction is potently inhibited by NADPH, indicating that the NAD⁺ : NADPH ratio in cells will determine whether AKR1C2 makes 5-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17-HSD activity, which eliminated 3-diol as androsterone. PC-3 cells expressed RoDH like 3-HSD and 11-cis-retinol dehydrogenase but these endogenous enzymes did not oxidize 3-diol to 5-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells high levels of AKR1C2 transcripts were detected in prostate cancer but not in cells from normal prostate. Thus in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH, or because it cannot surmount the oxidative 17-HSD present. Neither AKR1C2, RoDH nor 11-cis retinol dehydrogenase are a source of 5-DHT in PC-3 cells.