Background: Increased oxidative stress has been suggested as one of the underlying mechanisms in iodide excess-induced thy roid disease. Metallothioneins (MTs) are regarded as scavengers of reactive oxygen species (ROS) in oxidative stress. Our aim is to investigate the effects of propylthiouracil (PTU), a thyroid peroxidase inhibitor, perchlorate (KClO4), a competitive inhibitor of iodide transport, and thyroid stimulating hormone (TSH) on mitochondrial superoxide production instigated by high concentra tions of iodide in the thyroids of MT-I/II knockout (MT-I/II KO) mice. Methods: Eight-week-old 129S7/SvEvBrd-Mt1 tm1Bri Mt2tm1Bri/J (MT-I/II KO) mice and background-matched wild type (WT) mice were used. Results: By using a mitochondrial superoxide indicator (MitoSOX Red), lactate dehydrogenase (LDH) release, and methyl thia zolyl tetrazolium (MTT) assay, we demonstrated that the decreased relative viability and increased LDH release and mitochon drial superoxide production induced by potassium iodide (100 μM) can be relieved by 300 μM PTU, 30 μM KClO4, or 10 U/L TSH in the thyroid cell suspensions of both MT-I/II KO and WT mice (P<0.05). Compared to the WT mice, a significant de crease in the relative viability along with a significant increase in LDH release and mitochondrial superoxide production were de tected in MT-I/II KO mice (P<0.05). Conclusion: We concluded that PTU, KClO4, or TSH relieved the mitochondrial oxidative stress induced by high concentrations of iodide in the thyroids of both MT-I/II KO and WT mice. MT-I/II showed antioxidant effects against high concentrations of io dide-induced mitochondrial superoxide production in the thyroid.