Previously we reported that naloxone stereoisomers, in an opioid receptor-independent manner, attenuated the inflammation-mediated degeneration of dopaminergic neurons by inhibition of the activation of microglia, the resident immune cells in the brain. Recently we discovered that -amyloid peptide A (1-42) exhibited enhanced neurotoxicity toward both cortical and mesencephalic neurons through the activation of microglia and production of superoxide. The purpose of this study was to determine whether naloxone isomers had any effect on A (1-42)-induced neurodegeneration. Pretreatment of either cortical or mesencephalic neuron-glia cultures with 1 to 10 M (Ϫ)-naloxone, prior to treatment for up to 11 days with 0.1 to 3 M A (1-42), afforded significant neuroprotection as judged by neurotransmitter uptake, immunocytochemical analysis, and cell counting. More importantly, (ϩ)-naloxone, the ineffective enantiomer of (Ϫ)-naloxone in binding opioid receptors, was equally effective in affording neuroprotection. Mechanistically, inhibition of A (1-42)-induced production of superoxide in microglia underlay the neuroprotective effect of naloxone stereoisomers. Moreover, neuroprotection and inhibition of A (1-42)-induced superoxide production was also achieved with naloxone methiodide, a charged analog with quaternary amine, suggesting that the site of action for naloxone isomers is at the cell surface of microglia. These results demonstrated that naloxone isomers, through mechanisms unrelated to the opioid receptors, were capable of inhibiting A (1-42)-induced microglial activation and degeneration of both cortical and mesencephalic neurons. Combined with our previous observations with inflammagen-induced neurodegeneration, naloxone analogs, especially (ϩ)-naloxone, may have potential therapeutic efficacy for the treatment of Alzheimer's and Parkinson's disease.