The function of striatal adenosine A_2A receptors (A_2ARs) is well recognized because of their high expression levels and the documented antagonistic interaction between A_2ARs and dopamine D₂ receptors in the striatum. However, the role of extrastriatal A_2ARs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A_2ARs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A_2ARs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A_2AR knock-out (st-A_2AR KO) mice in comparison with forebrain-specific A_2AR KO (fb-A_2AR KO) mice. In contrast to fb-A_2AR KO (deleting A_2ARs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A_2AR KO mice exhibited Cre-mediated selective deletion of the A_2AR gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A_2AR KO but attenuated in fb-A_2AR KO mice. Furthermore, selective inactivation of the A_2ARs in extrastriatal cells by administering the A_2AR antagonist KW6002 into st-A_2AR KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A_2ARs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A_2ARs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A_2ARs.