The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.