Carotenoids are considered to be the first line of defense of plants against singlet oxygen (1O2) toxicity because of their capacity to quench 1O2 as well as triplet chlorophylls through a physical mechanism involving transfer of excitation energy followed by thermal deactivation. Here, we show that leaf carotenoids are also able to quench 1O2 by a chemical mechanism involving their oxidation. In vitro oxidation of β-carotene, lutein, and zeaxanthin by 1O2 generated various aldehydes and endoperoxides. A search for those molecules in Arabidopsis (Arabidopsis thaliana) leaves revealed the presence of 1O2-specific endoperoxides in low-light-grown plants, indicating chronic oxidation of carotenoids by 1O2. β-Carotene endoperoxide, but not xanthophyll endoperoxide, rapidly accumulated during high-light stress, and this accumulation was correlated with the extent of photosystem (PS) II photoinhibition and the expression of various 1O2 marker genes. The selective accumulation of β-carotene endoperoxide points at the PSII reaction centers, rather than the PSII chlorophyll antennae, as a major site of 1O2 accumulation in plants under high-light stress. β-Carotene endoperoxide was found to have a relatively fast turnover, decaying in the dark with a half time of about 6 h. This carotenoid metabolite provides an early index of 1O2 production in leaves, the occurrence of which precedes the accumulation of fatty acid oxidation products.