Superoxide radicals(O₂^-_·) produced in PSⅡ particles separated from spinach has been investigated by spin trapping-electron spin resonance (ESR) technique. After bubbling with oxygen and incubating with tetracyanoethylene (TCNE) that acts as an inhibitor of superoxide dismutase (SOD) the experimental evidence of spin adduct of ［DMPO-O₂H］^· can be obtained by in situ ESR measurements in PSⅡ particles. In contrast, adduct of O₂^-_· and DMPO produced under illumination is obviously decreased when SOD that usually performs as a scavenger of O₂^-_· is present. Furthermore, it is evident to find that the generation of O₂^-_· is positively correlated with the concentration of oxygen. The production of O₂^-_· is also pH dependent, and O₂^-_· concentration reaches maximum when pH is in the range of 6.0～6.5. Either high, or low pH value will lead it to descend steeply.In Tris-HCl washed PSⅡ particles donor-side ET inhibition can causes the production of O₂^-_· decreasing obviously. It can be concluded that the PSⅡ that is active in splitting water and oxygen generation is also a reactive site responsible for the photoinduced O₂^-_· generation in thylakoid of higher plant. In another word, the formation of O₂^-_· is relevantly correlated to the electron transport activity of PSⅡ.