The release of zinc (Zn) from glutamatergic synapses contributes to the neuropathology of ischemia, traumatic brain injury, and stroke. Astrocytes surround glutamatergic synapses and are vulnerable to the toxicity of Zn, which impairs their antioxidative glutathione (GSH) system and elevates the production of reactive oxygen species (ROS). It is not known whether one or both of these actions are the primary cause of Zninduced cell death in astrocytes. Using primary rat astrocyte cultures we have examined whether Zn-mediated impairment of GSH redox cycling is the main source of its toxicity. Zn acetate at concentrations of 100 ΞΌM or greater were found to inactivate glutathione reductase (GR) via an NADPH-dependent mechanism, while concentrations of 150 ΞΌM and above caused substantial cell death. Furthermore, Zn increased the ratio of GSSG:GSH in astrocytes, increased their export of GSSG, slowed their clearance of exogenous H 2 O 2 , and promoted the intracellular production of ROS. In contrast, the GR inhibitor, carmustine, did not induce cell death, cause the production of ROS, or alter the GSH thiol redox balance. Taken together these results indicate that Zn toxicity in astrocytes is primarily associated with the generation of intracellular ROS, rather than the inhibition of GR.