Methylmercury (MeHg) is a highly neurotoxic, environmentally ubiquitous chemical that exerts its toxic effects by largely unknown mechanisms. Maintenance of optimal intracellular concentrations of glutathione (GSH) is vital for cellular defenses against damage from free radicals. Since astrocytes play an essential role in providing GSH precursors to neurons, studies were directed at the effect of MeHg on cystine transport in both cell types. Astrocytes accumulated cystine via three independent transporters, referred to as system XAG-, system XC-, and gamma-glutamyltranspeptidase (GGT). In contrast, neurons accumulated cystine exclusively via system XC- and GGT. MeHg potently inhibited cystine uptake in astrocytes (but not in neurons), and this effect could be fully accounted for by inhibition of the system XAG- transporter. The transport of glutamate in astrocytes is also inhibited by reactive oxygen species (ROS). Accordingly, additional studies examined the ability of thiol reducing or oxidizing agents to inhibit the astrocytic transport of 3H-D-aspartate, a glutamate analog. The antioxidant catalase significantly attenuated MeHg-induced inhibition of astrocytic 3H-aspartate uptake. Combinedly, these studies suggest that inhibition of cystine uptake and decreased astrocytic GSH levels and efflux reduce the availability of precursors for GSH synthesis in neurons. In addition, MeHg-induced generation of H2O2 plays a role in the inhibition of astrocytic glutamate transport. These effects likely increase neuronal vulnerability to MeHg-induced oxidative stress, and excess N-methyl D-aspartate (NMDA) receptor activation leading to neuronal demise.