Glia undergo inflammatory activation in most CNS pathologies and are capable of killing cocultured neurons. We investigated the mechanisms of this inflammatory neurodegeneration using a mixed culture of neurons, microglia, and astrocytes, either when the astrocytes were activated directly with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) or LPS/IFN-γ-activated microglia were added to mixed neuronal cultures. In either case, activated glia caused 75–100% necrotic cell death within 48 hr, which was completely prevented by inhibitors of inducible nitric oxide synthase (iNOS) (aminoguanidine or 1400W). Activated astrocytes or microglia produced nitric oxide (NO) (steady-state level ∼0.5 μm), which immediately inhibited the cellular respiration of cocultured neurons, as did authentic NO. NO donors also decreased ATP levels and stimulated lactate production by neurons, consistent with NO-induced respiratory inhibition. NO donors or a specific respiratory inhibitor caused rapid (<1 min) release of glutamate from neuronal and neuronal–astrocytic cultures and subsequent neuronal death that was blocked by an antagonist of NMDA receptor (MK-801). MK-801 also blocked neuronal death induced by activated glia. High oxygen also prevented NO-induced neuronal death, consistent with death being induced by NO inhibition of cytochrome c oxidation in competition with oxygen. Thus activated glia kill neurons via NO from iNOS, which inhibits neuronal respiration resulting in glutamate release and subsequent excitotoxicity. This may contribute to neuronal cell death in inflammatory, infectious, ischemic, and neurodegenerative diseases.