In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.