Abstract Human aldo-keto reductase AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase) catalyzes the reduction of Δ 4-androstene-3,17-dione to yield testosterone, the reduction of 5α-dihydrotestosterone to yield 3α- and 3β-androstanediol, and the reduction of estrone to yield 17β-estradiol. Relatively, high mRNA expression of AKR1C3 was found in human prostate and mammary gland where it is implicated in regulating ligand access to the androgen and estrogen receptor, respectively. AKR1C3 shares high sequence identity >86% with related plastic human 20α-hydroxysteroid dehydrogenases (AKR1C1), type 3 3α-hydroxysteroid dehydrogenase (AKR1C2) and type 1 3α-hydroxysteroid dehydrogenase (AKR1C4), and reagents are urgently needed to discriminate between these enzymes at the mRNA, protein and functional level. We describe the characterization of a high-titer isoform specific monoclonal antibody (Ab) for AKR1C3. It does not cross react with human AKR1C1, AKR1C2 or AKR1C4, human aldehyde reductase AKR1A1 or rat 3α-hydroxysteroid dehydrogenase (AKR1C9) on immunoblot analysis. The monoclonal Ab can be used to detect AKR1C3 expression by immunohistochemistry in sections of paraffin-embedded mammary gland and prostate. In the breast enzyme staining was detected in ductal carcinoma in situ where the cancerous cells were strongly immunoreactive. In normal prostate immunoreactivity was limited to stromal cells with only faint staining in the epithelial cells. In adenocarcinoma of the prostate elevated staining was observed in the endothelial cells and carcinoma cells. The reagent thus has utility to access the localized expression of AKR1C3 in hormonal dependent malignancies of the breast and prostate.