Methamphetamine (METH) is a monoaminergic toxin that destroys dopamine terminals and causes astrogliosis in vivo. Oxidative stress has been shown to play an important role in the toxic effects of METH. In the present study, we sought to determine whether astrocytes are involved in METH-induced oxidative stress. Reactive oxygen species (ROS), ATP, and change in mitochondria membrane potential (ΔΨm) were examined in cultured striatal, mesencephalic, and cortical astrocytes after 4 to 48 h of 4 mM METH treatment. Results showed that only striatal and mesencephalic astrocytes showed a significant increase in ROS formation from 8 and 12 h, respectively. At 48 h treatment, there was a 55 and 53% increase in ROS content in striatal and mesencephalic astrocytes, respectively, whereas cortical astrocytes showed only a 25% (not significant) increase. JC-1, a ΔΨm-sensitive dye, showed a decrease in ΔΨm at 8 h treatment for striatal and mesencephalic astrocytes and at 12 h for cortical astrocytes. Astrocytes from all three regions showed a similar pattern of initial increase followed by a decrease in ATP content, with striatal astrocytes resulting in a maximum depletion (39% of control value) at 48 h treatment. These findings showed that METH treatment resulted in the formation of ROS in the order of striatal > mesencephalic > cortical astrocytes. Although the formation of ROS did not severely interfere with ATP production, a depolarization of mitochondria was observed. The present study suggested that astrocytes may be an important element governing the selective vulnerability to the striatum to METH-induced oxidative stress.