Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5β-cardenolides. Progesterone 5β-reductases (P5βRs) first described in Digitalis species are thought to be involved in 5β-cardenolide biosynthesis. P5βRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5βR was isolated from Erysimum crepidifolium leaves by 5'/3' RACE-PCR (termed EcP5βR). Subsequently, the P5βR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5' and 3' end primers deduced from the EcP5βR cDNA. The EcP5βR cDNA is 1170bp long and encodes for 389 amino acids. The EcP5βR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5βR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k(cat)K(M)⁻¹) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5βR was modelled using the crystal structure of P5βR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5βR-like enzymes were present and the protein sequence fitted into the template smoothly.