Calystegines are polyhydroxylated nortropanes which derive from the tropane alkaloid biosynthesis. Calystegine A3 and B2 accumulate as main alkaloids in the Convolvulaceae Calystegia sepium in all plant organs. Two cDNA sequences with high homologies to known tropinone reductases (TR) were isolated. Both sequences show typical motifs of the short chain dehydrogenase/reductase family. One cDNA was heterolog expressed in E.coli. Tropinone reduction by the enzyme led to formation of tropine exclusively, justifying the designation TRI. Transcript abundance of trI in all plant organs revealed a high transcription rate in root tissue, and a weak expression was seen in stem tissue. Investigation of the genomic structure of trI by Southern blotting indicates a gen family with minimum 3 different genes. The second sequence could be expressed neather in bacteria, nor in yeast or plants and is therefor designated as a putative TR. The native protein of the pseudotropine forming tropinone reductase (TRII) was isolated from root cultures, purified and characterised. It is similar in its biochemical properties to known TRII of the Solanaceae family. By application of 15N-labeled tropinone the biosynthesis of calystegines from tropinone over pseudotropine was prooved. The incorporation of label was analysed by NMR and quantified by GC-MS. Norpseudotropine was identified as a further metabolit of the calystegine biosynthesis.