From sterile root cultures of Datura stramonium, an NADP(H)-specific tropine dehydrogenase has been isolated and characterized. The enzyme catalyzes the reversible and stereospecific oxidation of tropine and related tropane-3 alpha-ols to the corresponding ketone. Isomeric pseudotropine (tropane-3 beta-ol) is neither accepted as substrate nor produced in the reverse reaction. It is assumed that this dehydrogenase is involved in the biosynthesis of tropane alkaloids.