The function of cytochrome b559 (cyt b559) in photosystem II (PSII) was studied in a tobacco mutant in which the conserved phenylalanine at position 26 in the β-subunit was changed to serine. Young leaves of the mutant showed no significant difference in chloroplast ultra structure or in the amount and activity of PSII, while in mature leaves the size of the grana stacks and the amount of PSII were significantly reduced. Mature leaves of the mutant showed a higher susceptibility to photoinhibition and a higher production of singlet oxygen, as shown by spin trapping electron paramagnetic resonance (EPR) spectroscopy. Oxygen consumption and superoxide production were studied in thylakoid membranes in which the Mn cluster was removed to ensure that all the cyt b559 was present in its low potential form. In thylakoid membranes, from wild-type plants, the larger fraction of superoxide production was 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive. This type of superoxide formation was absent in thylakoid membranes from the mutant. The physiological importance of the plastoquinol oxidation by cyt b559 for photosynthesis is discussed.