Plants have evolved various strategies to acclimate to high light conditions at different levels of organization. High light stress stimulates synthesis of different antioxidant enzymes and low molecular weight antioxidants, mainly in chloroplasts. In the present studies we showed that plastoquinol, in addition to alpha-tocopherol, is the main lipid-soluble antioxidant synthesized during acclimation of Arabidopsis plants to high light conditions. The level of plastoquinol increased >10-fold and independently of tocopherols, as revealed using tocopherol biosynthetic mutants. The high light-induced increase in plastoquinol level was mainly attributable to the photochemically non-active fraction of this compound localized in plastoglobuli, which are the storage site of prenyllipids for their antioxidant action. Our data also revealed that tocopherol cyclase is required for plastochromanol biosynthesis from plastoquinol in vivo. Plastochromanol accumulated in increasing amounts in leaves during growth and it was also identified in seeds. The obtained data suggest that plastochromanol may, similarly to other prenyllipids, fulfill antioxidant function in leaves and seeds, especially during aging.