Growing evidence implicates microglia in the loss of dopaminergic neurons in Parkinson's disease (PD). However, factors mediating microglial activation in PD are poorly understood. Proinflammatory cytokines, such as interferon-γ (IFN-γ), orchestrate the actions of microglia. We report here that PD patients express significantly elevated levels of IFN-γ in their blood plasma. After this initial finding, we found that IFN-γ-deficient mice displayed attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced substantia nigra pars compacta dopaminergic cell loss along with reduced loss of striatal tyrosine hydroxylase and dopamine transporter fiber density. MPTP-induced depletion of striatal dopamine and its metabolite DOPAC (3,4-dihydroxyphenylacetic acid), as well as ΔFosB, a marker of postsynaptic dysfunction, were also attenuated in these knock-out mice. Consistent with the role for IFN-γ in microglial activation, MPTP-induced morphological activation of microglia was abrogated compared with wild-type mice. To examine more mechanistically the role of IFN-γ in microglial activation, we evaluated the interactions between microglia and dopaminergic neurons in an in vitro mixed microglia/midbrain neuron rotenone-induced death paradigm. In this in vitro paradigm, dopaminergic neurons are selectively damaged by rotenone. Exogenous IFN-γ ligand alone and without rotenone resulted in dopaminergic cell loss, but only in the presence of microglia. The addition of an IFN-γ neutralizing antibody attenuated neuronal loss as a result of rotenone treatment. The presence of only wild-type microglia and not those deficient in IFN-γ receptor elicited significant dopaminergic cell loss when exposed to rotenone. Neurons deficient in IFN-γ receptor, however, did not display increased resistance to death. Finally, levels of IFN-γ message increased in microglia in response to rotenone. Together, these data suggest that IFN-γ participates in death of dopaminergic neurons by regulating microglial activity.