Abstract The generation of violaxanthin (Vx) de-epoxidase (VDE), photosystem II subunit S (PsbS) and zeaxanthin (Zx) epoxidase (ZEP) (VPZ) lines, which simultaneously overexpress VDE, PsbS and ZEP, has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the overexpression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3- to 5-fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of Vx to Zx, and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (antheraxanthin to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.