Manganese (Mn) is known to induce mitochondrial dysfunction in excessive dose; however the mechanisms underlying its action are not elucidated clearly. To determine if Mn 2 + can act directly on mitochondria or indirectly by producing reactive oxygen species (ROS), isolated mitochondria were exposed to different concentration of Mn 2 + (5, 50, 500, 1000 μM). ROS generation, respiratory control ratio (RCR), mitochondrial membrane potential (MMP) and respiratory chain complexes activities were investigated. Dose-dependent inhibition of respiratory chain complexes and induction of ROS were observed; these changes were paralleled by decreasing of respiratory control ratio (RCR) both with succinate or glutamate+malate. Further investigation indicated that the membrane potential determined by Rhodamine123 release decreased after MnCl 2 exposure at 1000 μM. In addition, effects of the antioxidants NAC (500 μM), GSH (500 μM) and Vitamin C (500 μM) were studied at 500 μM Mn 2 + . The results indicate that the effect of Mn 2 + exposure on respiratory chain is not site-specific, and antioxidants can protect the mitochondria function by reducing the formation of free radicals.