The amyloid β-peptide (Aβ) activates microglia and promotes the generation of cytokines and oxygen species, including nitric oxide (NO) and tumor necrosis factor α (TNF-α), which can be either neurotoxic or neuroprotective. We show that neuron death in cocultures of rat cortical microglia and neurons activated by lipopolysaccharide (LPS) or Aβ_1–42 plus interferon γ (IFNγ) is caused by short-lived diffusible molecules and follows the generation of superoxide and/or peroxynitrite as determined by electron paramagnetic spectroscopy. Neurotoxicity induced by LPS or Aβ_1–42 plus IFNγ is blocked by inhibitors of NO synthesis and by the peroxynitrite (ONOO⁻) decomposition catalysts FeTMPyP [5,10,15,20-tetrakis(n-methyl-4′-pyridyl)porphinato iron (III) chloride] and FeTPPS [5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride] but not by the TNF-α inhibitor pentoxifylline. The specificity of FeTMPyP for ONOO⁻ was confirmed by its ability to block the toxicity of a peroxynitrite donor but not of NO donors or of high levels of superoxide in a yeast mutant lacking superoxide dismutase 1. These results implicate peroxynitrite as a mediator of the toxicity of activated microglia, which may play a major role in Aβ_1–42 neurotoxicity and Alzheimer's disease.