Pesticide exposure, such as rotenone or paraquat, increases the risk of Parkinson's disease. It is established that the inhibition of mitochondrial complex I is involved in rotenone-induced dopaminergic neurotoxicity. However, the mechanism underlying selective dopaminergic neurotoxicity by rotenone exposure remains unknown. Recently, we demonstrated astrocyte-microglia interaction promoted rotenone-induced non-cell-autonomous dopaminergic neurodegeneration. In this study, we examined involvement of region-specific glial crosstalk in rotenone neurotoxicity. We prepared mesencephalic neuronal culture and glial cell culture (astrocyte+microglia) from mesencephalon or striatum of SD rats embryos at 15 days of gestation. Direct treatment of mesencephalic neuronal cultures with rotenone failed to decrease dopaminergic neurons. Dopaminergic neurotoxicity was induced by treatment with conditioned media from rotenone-treated mesencephalic, but not striatal, glial cells. Furthermore, level of an antioxidant metallothionein-1 was reduced in the conditioned media from mesencephalic, but not striatal, glial cells following rotenone treatment. These results suggest that region-specific astrocyte-microglia interaction could play an important role in rotenone-induced dopaminergic neurodegeneration.