3-Methylcatechol (3MC) is a metabolite of toluene. 3MC spontaneously oxidizes in physiological medium consuming oxygen and forming reactive oxygen species and quinones. AIM: To clarify whether 3MC could be toxic towards the central nervous system, we examined the effects of 3MC on mitochondrial basal respiration in organelles obtained from rat brains, and we also tested the cytotoxicity towards rat primary astrocytes and human glioblastoma GL-15 cells in vitro. METHODS: Mitochondria were isolated from brains of adult rats by differential centrifugation. Sodium succinate was added to a final concentration of 10 mM in order to induce mitochondrial basal respiration (state 2) and oxygen consumption was carried out at 37 °C in a closed chamber containing a Clark type oxygen electrode connected to a monitor. Cultures of isolated cortical astrocytes were prepared from the neocortex of 1-day-old Wistar rat brains, maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 IU/ml), and streptomycin (100 mg/ml). GL-15 cells were seeded in 96-well plates to obtain a final density of 3 ´ 10⁴ cells/cm² and they were maintained in the same medium described above supplemented with 5% glucose.. Logarithm dilutions of 3MC in the range of 60 to 3,000 mM were used to examine the cytotoxic effect of 3MC towards astrocytes and GL-15 cells in order to determine the EC₅₀. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: This compound significantly inhibited state 2 FADH₂-linked mitochondrial respiration. Furthermore, this toluene metabolite was cytotoxic towards rat primary astrocytes and GL-15 cells. The concentration that killed 50% of astrocytes after 72 hours was 107 mM. The concentration that killed 50% of GL-15 cells was 641 mM. CONLUSIONS: The results of this study indicate that 3MC inhibits FADH₂-linked respiration. This molecule was toxic towards rat astrocytes and human glioblastoma GL-15 cells. However, glioblastoma cells were more resistant than primary astrocytes. (Supported by CNPq, FUNDECI, FAPESB).