During senescence, production of reactive oxygen species increased in thylakoids. In two barley varieties, no difference in superoxide production was observed while singlet oxygen production increased only in one variety. During senescence, chlorophyll content decreased and photosynthetic electron transport was inhibited as shown for flag leaves collected from barley varieties Lomerit and Carina grown in the field. Spin trapping electron paramagnetic resonance (EPR) was used to investigate the production of reactive oxygen species in thylakoid membranes during senescence. EPR measurements were performed with specific spin traps to discriminate between singlet oxygen on one hand and reactive oxygen intermediates on the other hand. The results show that the generation of reactive oxygen intermediates increases in both varieties during senescence. Singlet oxygen increased only in the variety cv. Lomerit while it remained constant at a low level in the variety cv. Carina. Measurements in the presence of inhibitors of photosystem II and of the cytochrome b6f complex revealed that in senescing leaves reduction of oxygen at the acceptor side of photosystem I was the major, but not the only source of superoxide anions. This study shows that during senescence the production of individual reactive oxygen species varies in different barley varieties.