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This paper presents a detailed analysis of thylakoids isolated from secondary barley leaves harvested 18, 22, 25, 29, 32, 35 and 39 days after sowing (DAS). Goal of the analysis was to investigate the production of different reactive oxygen species (ROS) during development and senescence of barley. Generation of superoxide anion (Oradical dot₂−) and hydrogen peroxide (H₂O₂) increases during development of barley reaching the highest value right after the onset of senescence (between 25 and 29 DAS), thereafter the levels of both ROS start to decrease until 35 DAS when production of H₂O₂ increases again. In comparison with Oradical dot₂− and H₂O_2, generation of singlet oxygen (¹O₂) showed continuous production of low amounts thought the duration of experiment. Oxidative damage to the thylakoid membrane was assessed by measuring lipid peroxidation. Results showed gradual increase in lipid …