AbstractThe function of astrocytes in cerebral cortex may be studied by comparing the properties of conventional, astrocyte‐rich cultures with astrocyte‐poor cultures in which astrocyte proliferation has been stringently suppressed. Exposure of astrocytepoor, but not astrocyte‐rich, cultures to fresh medium containing 2 mM glutamine resulted in the death of most neurons within 24 h. This study was undertaken to understand the basis for the apparent toxicity of glutamine in astrocyte‐poor cultures. The toxicity of glutamine was found to be mediated by glutamate, which demonstrated an LD50 as a neurotoxin in astrocyte‐poor cultures of 2 μM. Exposure of astrocyte‐poor (but not astrocyte‐rich) cultures to fresh medium containing glutamine for 17.5–24 h resulted in the accumulation of substantial quantities of glutamate (255 ± 158 μM; mean ± standard deviation) coincident with the death of neurons in the cultures. Exposure of astrocyte‐poor cultures to glutamate in the absence of glutamine did not result in the accumulation of extracellular glutamate. Both the neuronal death and the extracellular glutamate accumulation in astrocyte‐poor cultures exposed to glutamine could be blocked by N‐methyl‐D‐aspartate (NMDA) antagonists. These observations suggest that astrocytes as well as glutamine may play an important role in the pathogenesis of glutamate neurotoxicity