Methamphetamine, a psychostimulant drug, produces both acute psychomotor stimulation and long-lasting behavioral effects including addiction and psychosis. To identify anatomical substrates for the discriminative stimulus effects of methamphetamine in rats, we examined the drug discrimination-associated c-Fos expression in the brains of rats that were trained to discriminate methamphetamine from saline under a two-lever fixed ratio (FR-20) schedule of food reinforcement. c-Fos expression in the brains of rats trained to discriminate methamphetamine from saline was significantly increased in the nucleus accumbens (NAc) and the ventral tegmental area (VTA) as compared with the expression in the control rats that were maintained under the FR-20 schedule, but no alternation was observed in other areas including the cerebral cortex, caudate putamen, substantia nigra, hippocampus, amygdala, and habenulla. Methamphetamine treatment in the trained rats caused a significant increase in c-Fos expression in the VTA, and a decrease in the NAc core, as compared to saline treatment. However, c-Fos expression in the NAc and VTA of rats that received chronic intermittent methamphetamine administration without discrimination training, did not differ from the expression in saline-treatment animals. These results suggest that the VTA and the NAc play an important role in the discriminative stimulus effects of methamphetamine.