The neurotoxin 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP), depletes dopaminergic neurons in the substantia nigra, leading to symptoms of Parkinsonism. MPTP is used in the animal models of Parkinson’s disease where it destroys dopaminergic cells mainly in the substantia nigra, leading to depletion of dopamine in the striatum. Among the proposed mechanisms for nigral neuronal death is oxidative stress and mitochondrial dysfunction. Astrocytes play a key role in clearing potentially toxic byproducts of metabolism as well as in the process that converts the MPTP to its toxic form 1-methyl-4-phenylpyridinium (MPP+), which acts by interfering with complex I of the mitochondrial electron transport chain (Sayre, 1989). By measuring changes in mitochondrial membrane potential (ΔΨ), we sought to establish if astrocytes in the mesencephalon are more susceptible to MPTP toxicity than astrocytes from other brain areas. Astrocytes were cultured from the ventral mesencephalon, striatum and cortex of neonatal C57BL/6 mice. Cells were subcultured onto 25mm coverslips and incubated at 37°C in 5% CO2 to form confluent monolayers. Cells were then treated with 500 µM MPTP for 4, 8 or 24 hours. To measure ΔΨ, cells were washed in normal HBSS and then incubated in 10 µM JC-1 for 15 minutes. JC-1 (5,5’6,6′- tetraethylbenzimidazolecarbocyanine iodide) is a ratiometric dye that accumulates in mitochondria and forms J-aggregates when membrane potential is high (normal) and remains in a monomeric form when membrane potential is low (Reers el al., 1995). Using confocal microscopy (Biorad MRC 1024) and a 63x high NA objective, a 488 nm argon ion laser line was used for excitation while emission was simultaneously collected at 522 ± 35 nm and 585 nm corresponding to peak fluorescence from the monomer (527 nm) and J-aggregate (590 nm) of JC-1 respectively. Offline image processing was carried out to combine the green and red channels of the saved confocal image stacks. Our results showed that compared to controls (with no MPTP treatment), cortical astrocytes only showed significant dissipation of ΔΨ after 24 hours. Striatal astrocytes’ mitochondria were still highly polarized after 24 hours of exposure to MPTP. Mesencephalic astrocytes were the most affected by MPTP; within 4 hours, there was a greater degree of ΔΨ collapse than was seen in striatal astrocytes after 24 hours of treatment. By 24 hours, all mesencephalic astrocytes showed near complete dissipation of ΔΨ. We conclude that astrocytes cultured from different brain regions differ in their susceptibility to MPTP toxicity in the order mesencephalon > cortex > striatum.