The purpose of this study was to assess and compare the toxicity of β-amyloid (Aβ) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Aβ-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron–glia cultures with Aβ(1–42) over the concentration range 0.1–6.0 μm caused concentration-dependent neurotoxicity. High concentrations of Aβ (6.0 μm for cortex and 1.5–2.0 μm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Aβ (1.0–3.0 μm for cortex and 0.25–1.0 μm for mesencephalon) caused significant neurotoxicity in mixed neuron–glia cultures, but not in neuron- enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Aβ, including the observations that low concentrations of Aβ activated microglia morphologically in neuron–glia cultures and that addition of microglia to cortical neuron–glia cultures enhanced Aβ-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron–glia cultures. Low doses of Aβ significantly increased the production of superoxide anions, but not of tumor necrosis factor-α, interleukin-1β or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Aβ toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Aβ-induced neurotoxicity in neuron–glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Aβ-induced neurotoxicity was further substantiated by a study which showed that Aβ caused less of a decrease in dopamine uptake in mesencephalic neuron–glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Aβ is via the production of reactive oxygen species.