Paraquat (1,1'-dimethyl-4,4'-bipyridinium, PQ) is a herbicide to possibly induce Parkinson's disease (PD), since a strong correlation has been found between the incidence of the disease and the amount of PQ used. In this study, we examined PQ toxicity in rat organotypic midbrain slice cultures. PQ dose dependently reduced the number of dopaminergic neurons in cultured slices. Since this damage was prevented by GBR-12909, the dopamine transporter could be an initial step of the PQ induced dopaminergic neurotoxicity. The sequential treatments with lower PQ and 1-methyl-4-phenyl pyridinium (MPP+) doses, where each dose alone was not lethal, markedly killed dopamine neurons, suggesting that the exposure of a lower dose of PQ could lead to the vulnerability of dopaminergic neurons. This cell death was prevented by the inhibitors of NMDA, nitric oxide synthase (NOS), cycloheximide and caspase cascade. Neurons expressing NOS were identified inside and around the regions where dopamine neurons were packed. The cell death induced by the sequential treatments with PQ and MPP+ was also rescued by L-deprenyl and dopamine D2/3 agonists. These results strongly support that the constant exposure to low levels of PQ would lead to the vulnerability of dopaminergic neurons in the nigrostriatal system by the excitotoxic pathway, and might potentiate neurodegeneration caused by the exposure of other substances and aging.