Macrophages have been found to release glutamate and thereby induce neuronal cell death by excitotoxicity, a mechanism that seems to be operative in various neurologic diseases. In this study, it is shown that the presence of both cystine and glutamine in the culture medium is indispensable for brain macrophages to release glutamate and to cause neuronal cell death. Furthermore, release of glutamate requires protein synthesis since cycloheximide prevented accumulation of the neurotoxic molecule in supernatants of microglial cell cultures. Aminoadipate, which was shown to inhibit the uptake of cystine by system xc- in fibroblasts, efficiently reduced the release of glutamate. The requirement of glutamine and cystine for the release of glutamate by microglial cells as well as the inhibitory effect observed with aminoadipate shows the transport system xc- to be essential for the release of the excitotoxin glutamate by microglial cells. Phagocytosis of zymosan particles and stimulation with different bacterial components, such as LPS, protein A, tuberculin, and Staphylococcus enterotoxin A increased glutamate release two- to threefold above basal values. In addition, the effect of bacterial components was mimicked by TNF-alpha, but not by IL-1 and IL-6. Cytokines known to deactivate macrophages, such as TGF-beta, IL-4, and IL-10, did not affect the transport system xc- in microglial cells. However, dexamethasone suppressed the glutamate release up to 50%.