Abuse of methamphetamine (MAP) and cocaine causes severe medical and social problems throughout the world. Our previous study found that trihexyphenidyl (THP), a muscarinic cholinergic receptor antagonist, specifically suppressed the rewarding properties of MAP but not of cocaine, as measured by conditioned place preference in mice. The present study examined using in vivo microdialysis whether THP differentially affects the extracellular dopamine (DA) levels in the nucleus accumbens and striatum of mice injected with MAP and cocaine in comparison with another antimuscarinic agent, scopolamine (SCP). In addition, locomotor activity was simultaneously measured during microdialysis. In vivo microdialysis experiments revealed that during the initial hour after injection of MAP (1 mg/kg) DA levels increased up to 698% in the nucleus accumbens and 367% in the striatum as compared to the basal level. These increases were reduced to 293% in the nucleus accumbens and 207% in the striatum by treatment with 5 mg/kg THP. However, SCP (3 mg/kg) had no effect on the increases in extracellular DA levels in both regions after MAP injection. Cocaine (10 mg/kg) increased DA levels during the initial hour to 254% in the nucleus accumbens and 220% in the striatum as compared to the basal level. These increases were unaffected by treatment with either THP or SCP. On the contrary, both THP and SCP enhanced the locomotor-stimulant action of MAP and cocaine. These results, together with our previous finding, suggest that THP may specifically antagonize the rewarding properties of MAP through suppression of DA release in the mesolimbic area without retarding locomotor activity. Synapse 49:47–54, 2003. © 2003 Wiley-Liss, Inc.