Protein extracts prepared from Armoracia rusticana leaves exhibited progesterone 5β-reductase activity (P5βR). Using primers deduced from known P5βR genes a full-length cDNA encoding a progesterone 5β reductase (ArP5βR, EC 1.3.99.6) was isolated from Armoracia rusticana leaves. A SphI/SalI ArP5βR PCR fragment was cloned into the pQE30 vector and transformed into Escherichia coli. The recombinant His-tagged fusion protein gene was expressed in E. coli M15. Progesterone was enantio selectively reduced to 5β-pregnane-3,20-dione. The ArP5βR of A. rusticana (JF460011) showed 92 and 91% sequence identity to steroid 5β-reductases isolated from Arabidopsis thaliana and Erysimum crepidifolium, respectively. A three-dimensional model of ArP5βR on the basis of the Digitalis lanata P5βR (PDB ID: 2V6G, DlP5βR) highlights the close structural relationship to Vein Patterning 1-encoded enone reductases described previously. Key message. Further evidence is provided that VEP1-encoded enone reductases (progesterone 5β-reductases; P5βR) are distributed ubiquitously in the angiosperms and that VEP1 may be used as a molecular marker to infer taxonomic relationship in Brassicaceae.