Methamphetamine is a powerfully addictive psychostimulant that dramatically affects the mammalian central nervous system. Methylphenidate has been shown to have psychostimulus effects similar to methamphetamine. In the present study, we compared several effects of methylphenidate to those of methamphetamine. The subcutaneous administration of either methamphetamine or methylphenidate increased extracellular dopamine levels in the nucleus accumbens of mice. Interestingly, methamphetamine, but not methylphenidate, also increased the extracellular serotonin levels in this area. Further, repeated treatment with methamphetamine induced the development of sensitization to hyperlocomotion, whereas methylphenidate failed to induce behavioral sensitization. Moreover, in vitro treatment with methamphetamine, but not methylphenidate, caused long-lasting astrocytic activation in limbic neuron/glia co-cultures. These findings suggest that, unlike methamphetamine, methylphenidate shows a lack of development of behavioral sensitization to its hyperlocomotion and induces reversible astrocytic activation.