Methylmercury (MeHg) is an environmental contaminant that continues to cause risk to human health. The toxic effects of MeHg on the CNS implicate the involvement of glutamatergic system. In this study, we evaluated the effects of MeHg on [3H]glutamate uptake by synaptic vesicles. MeHg inhibited [3H]glutamate uptake in a concentration dependent manner. Since glutamate uptake by synaptic vesicles is driven by an electrochemical gradient, formed across the vesicle membrane by a bafilomycin A(1)-sensitive H+-ATPase, we further investigated the effect of MeHg on activity of this enzyme. MeHg inhibited the H+-ATPase activity and also dissipated the proton gradient (DeltapH), indicating that MeHg decreased [3H]glutamate uptake involving the H+-ATPase activity. Until now, the toxic effects of MeHg on CNS were attributed mainly to an impairment of glial glutamate transporters. These findings contribute for the understanding of the neurotoxicity by MeHg, pointing to the involvement of vesicular glutamate.