In chloroplasts of plants the xanthophyll cycle is suggested to function as a protection mechanism against photodamage. Two enzymes catalyze this cycle. One of them, violaxanthin de-epoxidase, transforms violaxanthin (Vio) to zeaxanthin (Zea) via antheraxanthin (Anth) and is bound to the lumenal surface of the thylakoid vesicles, when being in its active state. The other enzyme, Zea-epoxidase, is responsible for the backward reaction (Zea-->Anth-->Vio) and is active at the stromal side of the thylakoid. For the epoxidation of Zea this enzyme requires NAD(P)H and O2 as cosubstrates. Using isolated thylakoid membranes we found that FAD enhances the epoxidase activity (decrease of apparent Km for NAD(P)H and two-fold increase of Vmax). The flavin functions as a third cofactor which is partially lost during the isolation procedure of thylakoids. Other flavins, such as FMN or riboflavin are without effect. The involvement of FAD in the enzymatic reaction is also demonstrated by the inhibitory action of diphenyleneiodoniumchloride (DPI) (IC50 = 2.3 microM), a compound that blocks the reoxidation of reduced flavins within enzymes. The Zea-epoxidase is a multi-component enzyme system which can be classified as FAD-containing, NAD(P)H- and O2-dependent monooxygenase that is able to epoxidize 3-hydroxy beta-ionone rings of xanthophylls in the 5,6 position.