This study evaluated the role of thrombin-activated microglia in the neurodegeneration of mesencephalic cultures. Immunocytochemical and biochemical evidence indicated that in co-cultures consisting of rat cortical microglia and mesencephalic neurons, thrombin led to nonselective loss of mesencephalic neurons. Accompanying neurodegeneration, microglial activation was obvious, evidenced by expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and by increasing production of TNF-alpha and nitric oxide (NO). In mesencephalic neurons treated with conditioned media (CM) taken from thrombin-activated microglia, the number of dopaminergic neurons was significantly attenuated. The neurotoxicity of the CM was diminished when it was derived from microglia co-treated with thrombin and either an extracellular signal-regulated kinase 1/2 (ERK1/2) pathway inhibitor (PD98059) or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB203580). Moreover, jun N-terminal kinase (JNK) and p38-MAPK were activated in mesencephalic neurons treated with CM of thrombin-activated microglia. Inhibition of JNK and p38-MAPK rescued the dopaminergic neurons. Collectively, these results indicate that thrombin-activated microglia induce neurodegeneration in cultured mesencephalic neurons and that the MAPKs actively participate in both microglial activation and neurodegeneration. The present data carefully suggest that microglial activation triggered by thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.