The generation of superoxide anion radical (O2.-) in the cytochrome b6f complex (Cyt b6f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O2.- was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN3 and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O2.-, which is produced under high-light illumination of the Cyt b6f from spinach, might be generated from a reaction involing 1O2, and the Rieske Fe-S protein could serve as the electron donor in the O2.- production. The mechanism of photoprotection of the Cyt b6f complex by antioxidants is discussed. and M. Sang ... [et al.].