Enzymatic conversion of 24-epiteasterone to 3-dehydro-24-epiteasterone, representing a reversible step in the biosynthesis of 24R-methyl brassinosteroids, was monitored in vitro in Arabidopsis thaliana and Lycopersicon esculentum using fluorescent tagging and HPLC analysis. In both species 24-epiteasterone was metabolized by the cytosolic fraction of induced root-callus suspension cultures in a NAD dependent manner. This 3beta-dehydrogenation was only slightly inhibited by 12-fold excess of other 3beta-hydroxy intermediates of the early (cathasterone) and late C-6 oxidation pathway (6-deoxo-cathasterone, 6-deoxo-teasterone), indicating a rather specific protein. 3-Dehydro-6-deoxoteasterone and 3alpha-hydroxy intermediates of brassinosteroid biosynthesis did not reduce conversion of 24-epiteasterone to 3-dehydro-24-epiteasterone. In contrast to light-grown cultures the reaction was clearly inhibited in the dark.