The physiological function of microglial glutamate uptake has been debated as it is about 10% of that measured for astrocytes. This study addresses how glutamate, taken up from the extracellular space, is utilized by microglia. It was found that purified rat microglia incubated for 60 min with 3H-glutamate had an increased intracellular accumulation of 3H-glutamate after 12 h incubation with tumour necrosis factor alpha (TNF-α) but not after incubation with lipopolysaccharide (LPS). Furthermore, LPS- but not TNF-α-treated cells showed an increased efflux of 3H-labelled compounds, presumably glutamate through the XC– system and treatment with LPS or TNF-α increased the microglial glutathione concentrations and led to an increased incorporation of 3H-glutamate into glutathione. Depending on the stimuli, 3–6% of the total labelled contents were found in the form of glutathione and 25–35% in the form of glutamate. These results show that microglial glutamate uptake is directly coupled to glutathione synthesis and release of glutamate and/or glutamate metabolites. Additionally, the increased glutathione contents after LPS or TNF-α treatment were able to reduce microglial cell death after H2O2 challenge, showing a potential (self)-protective function for microglial glutamate transporter expression and glutathione synthesis.