The Arabidopsis thaliana VEP1 gene product shows about 70% sequence identity to Digitalis lanata progesterone 5beta-reductase, an enzyme considered to catalyze a key step in the biosynthesis of cardiac glycosides. A. thaliana does not accumulate cardenolides but protein extracts prepared from its leaves were capable of reducing progesterone to 5beta-pregnane-3,20-dione. A full-length cDNA clone encoding a Delta(4,5)-steroid 5beta-reductase (At5beta-StR, EC 1.1.1.145/1.3.1.23), a member of the short-chain dehydrogenase/reductase (SDR) family, was isolated from A. thaliana leaves. A SphI/SalI At5beta-StR gene fragment was cloned into the pQE vector system and transformed into Escherichia coli. The gene was functionally expressed and the recombinant His-tagged fusion protein was characterized. K(m) values and specific activities for putative 3-oxo-Delta(4,5)-steroid substrates such as progesterone, cortisol, cortexone and 4-androstene-3,17-dione, and for the co-substrate NADPH were determined. Progesterone was stereo-specifically reduced to 5beta-pregnane-3,20-dione and none of the 3-oxo-Delta(5,6)-steroids tested were accepted as a substrate. The gene encoding At5beta-StR was strongly transcribed in stems and leaves. A three-dimensional model of At5beta-StR highlights a close structural similarity to the related, previously described D. lanata progesterone 5beta-reductase. This homology extends to the active site where single amino acid substitutions might be responsible for the increased catalytic efficiency of At5beta-StR when compared to the activity of the recombinant form of the D. lanata enzyme.