3β-Hydroxysteroid dehydrogenases (3β-HSD) catalyze the dehydrogenation of 3β-hydroxyl groups of steroids into 3-oxo groups or, in reverse direction, the reduction of 3-oxo groups. In procaryotes they are involved in steroid metabolism in terms of detoxification and assimilation. Mammalian 3β-HSDs are bifunctional enzymes with an additional ketosteroid isomerase activity (KSI) and play an important role in the biosynthesis of steroid hormones. About the function of 3β-HSDs in steroid metabolism in the plant kingdom is little known so far. In the present work, potential 3β-HSD candidates showing high sequence homology to Δ5-3β-HSD (EC 220.127.116.11) from Digitalis lanata, which is involved in pregnane formation during cardenolide biosynthesis, were isolated from an Arabidopsis thaliana cDNA library and heterologously expressed. The most attractive candidates ‒ namely AtHSD1 (At2g47140) and AtHSD2 (At2g47130) ‒ were homogeneously purified and kinetically characterized. They can be assigned to the short-chain dehydrogenase / reductase family (SDR) and possess a wide substrate specificity for 3β-hydroxysteroids and 3-ketosteroids, respectively. They were quite tolerant in terms of variations of the sterol nucleus and side-chain structures. C19-, C21-, C27-, C28- und C29- steroids, as well with a Δ5-double bond in the B-ring as without one, were dehydrogenated and Δ5-, 5α- or 5β-configurated ketosteroids were reduced. For AtHSD1 a KSI activity could be demonstrated. In contrast to the enzyme from Digitalis, the enzymes from Arabidopsis showed higher substrate affinities for 5α-configurated steroids. Hypothetically an involvement in brassinosteroid biosynthesis was supposed. The conversion of the brassinosteroid precursors campesterol and cholesterol, and of a teasterone analogon 28-homoteasterone could be demonstrated. T-DNA mutants with insertions in the regions of the gene loci of AtHSD1 and AtHSD2 showed partially a growth-deficient phenotype with delayed germination and development. It could be concluded that the physiological role of AtHSD1 is mainly constricted to the root and that AtHSD2 plays a major role for the development of the whole plant. Further investigations should focus on the quantitative and qualitative analysis of the endogenous content of brassinosteroids in the mutants in order to ascertain the plausible function in the brassinosteroid biosynthesis.