Abstract The aim of the present study was to evaluate the production of superoxide anion (O2− ) in the trigeminal complex nuclei after a functional mechanical overload of the teeth due to the preference for masticating on one side in rats. The preference for masticating on one side was induced by the discomfort due to a small abrasion of one molar; such lateralisation in mastication was confirmed by electromyography. The production of O2− was evaluated in the trigeminal nuclei by fluorescence microscopy after an injection of dihydroethidium. The results showed that there was an increased production of O2− in the subnucleus oralis of the spinal trigeminal nucleus in the same side where the mastication was preferred. This result demonstrates that an increased activity of non-painful sensory neurons can enhance the production of reactive oxygen species within the central second order sensory nuclei.