Methamphetamine (METH) has long-lasting neurotoxic effects on the dopamine and forebrain serotonin systems. It was reported that METH would induce the release of glutamate within the striatum and that it also caused astrogliosis. The mechanisms of this release and subsequent neurotoxicity are not well defined. The aim of this study was to examine the response of cultured astrocytes after METH-induced injury. Astrocytes were cultured from neonatal C57B1/6 mice brains. Cells were obtained from the mesencephalon, striatum and cortex in order to examine any regional differences. Cells were treated with 4mM METH for 4, 8, 12, 24 and 48 hr. Lactate dehydrogenase (LDH) levels were used as a measure of cell viability. At various time points, Western blot analyses were performed to study the change in GFAP and vimentin (markers for astrogliosis) levels. Change in glutamine synthase (GS), the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia in astrocytes, was also examined. The results showed that METH caused marked astrogliosis in striatal and mesencephalic astrocytes. Cells were transformed from protoplasmic (inactive) to fibrous (reactive) form after 48 hr treatment. There were also large amounts of vacuoles present in the cytoplasm of these cells. LDH results showed that there was only slight increase in enzyme levels after 48 hr treatment suggesting that the astrogliosis observed was not due to the decrease in cell viability. The amount of GS were depleted more rapidly in striatal astrocytes (50% of control by 8 hr treatment) followed by mesencephalic astrocytes (reaching 10% of control by 48 hr treatment). Cortical astrocytes showed only a 48% depletion by 48 hr treatment, indicating that they are more resistant to METH-induced toxicity. The rapid depletion of GS obtained in striatal and mesencephalic astrocytes suggested that astrocytes of the dopaminergic system are more sensitive to METH-induced injury. This may be due to the direct effects of METH-induced oxidative stress on the mitochondria of these cells resulting in GS depletion and astrogliosis.