Previous studies have demonstrated that there is an increase in oxidative stress in the cerebral cortex of rats after repeated painful stimulation and that long-lasting pain increases the production of superoxide ion (O(2) (-)), nitric oxide and peroxynitrite due to the activation of AMPA and NMDA receptors. The purpose of the present study was to evaluate the possible role of O(2) (-) in the transmission of oro-facial pain. Formaldehyde 1% was injected subcutaneously into one vibrissal pad of adult male Sprague-Dawley rats as a model of persistent pain, then O(2) (-) production and superoxide dismutase (SOD) activity were evaluated in the left and right spinal trigeminal nuclei. O(2) (-) production was revealed using dihidroetidium (DHE) injected at 10 or 45 min after the formalin injection in conscious or anaesthetized rats. A histochemical assay for SOD was performed to evaluate the activity of SOD at 10 min after the formalin injection. The results showed a significant increase in O(2) (-) production in the homolateral nucleus at 45 min. However, there was no significant difference between the two sides at 10 min after the formalin injection. No significant difference was observed in SOD activity between the two sides of the spinal trigeminal nucleus. This study demonstrated that there is an increased production of O(2) (-) in the second phase but not in the first phase of the formalin test; thus O(2) (-) is involved in pain induced by inflammation, but not in acute pain.