Protoporphyrinogen oxidase-inhibiting herbicides cause massive accumulation of protoporphyrin IX (Proto IX) in treated plant cells. Reactive oxygen species (ROS) induced by the accumulated Proto IX is thought to cause peroxidative damage, however, the process of peroxidation is still unclear. To investigate the mechanism of the peroxidation of lipids by Proto IX-induced ROS, we used an oxygen electrode. By measuring dissolved oxygen consumption in the reaction mixture, the rate of peroxidation was evaluated in vitro. Production of lipid hydroperoxide was confirmed by measuring thiobarbituric acid reacting substances (TBARS). The linear decrease of dissolved oxygen and linear increase of lipid hydroperoxides occurred immediately after light irradiation, suggesting that the method used in this study is applicable as a model system to investigate lipid peroxidation by Proto IX-induced ROS. Determination of the antioxidants effect on the reduction of dissolved oxygen indicated that both superoxide and hydrogen peroxide scavenging enzymes as well as singlet oxygen scavengers remarkably suppressed the peroxidative reaction. These results suggest that in addition to singlet oxygen, other ROS are involved in the Proto IX-induced peroxidation processes in plant cells. Furthermore, comparison of porphyrin intermediates derived from chlorophyll synthesis showed that the peroxidizing ability of Proto IX was not higher than other porphyries. This suggests that greater accumulation of Proto IX is necessary to initiate lipid peroxidation in plant cells.