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Abstract: Exposure to elevated levels of manganese has been shown to cause neuronal damage in the midbrain and the development of Parkinsonian symptoms. Activation of microglia and release of neurotoxic factors in particular free radicals are known to contribute to neurodegeneration. We have recently reported that manganese chloride (MnCl2) stimulates microglia to produce reactive oxygen species (ROS). The aim of this study is to determine the role of microglia in the MnCl2-induced degeneration of dopaminergic (DA) neurons that are particularly vulnerable to oxidative insult. MnCl2 (10–300 μM; 7 days) was markedly more effective in damaging DA neurons in the rat mesencephalic neuron–glia cultures than the neuron-enriched (microglia-depleted) cultures. In addition, the microglia-enhanced MnCl2 toxicity was found to be preferential to DA neurons. The microglial enhancement of DA neurotoxicity was further supported by the observation that replenishment of microglia to the neuron-enriched cultures significantly increased the susceptibility of DA neurons to the MnCl2-induced damage. Analysis of the temporal relationship between microglial activation and DA neurodegeneration revealed that MnCl2-stimulated microglial activation preceded DA neurodegeneration. Mechanistically, MnCl2 (10–300 μM) stimulated a concentration- and time-dependent robust production of ROS and moderate production of nitric oxide but no detectable release of tumor necrosis factor-alpha and interleukin-1beta. Application of free radical scavengers including superoxide dismutase/catalase, glutathione, N-acetyl cysteine and an inhibitor of nitric oxide biosynthesis significantly protected DA neurons against the MnCl2-induced degeneration. These results demonstrate that microglial activation and the production of reactive nitrogen and oxygen free radicals promote the MnCl2-induced DA neurodegeneration. [Copyright &y& Elsevier]