Evidence for heteromeric receptor complexes comprising adenosine A2A and metabotropic glutamate 5 (mGlu5) receptors in striatum has raised the possibility of synergistic interactions between striatal A2A and mGlu5 receptors. We investigated the role of striatal A2A receptors in the locomotor stimulant and antiparkinsonian properties of mGlu5 antagonists using complementary pharmacologic and genetic approaches. Locomotion acutely stimulated by the mGlu5 antagonist [2-methyl-6-(phenylethynyl)-pyridine (MPEP)] was absent in mGlu5 knock-out (KO) mice and was potentiated by an A2A antagonist KW-6002 [(E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine], both in normal and in dopamine-depleted (reserpinized) mice. Conversely, the MPEP-induced motor response was markedly attenuated in single and double A2A and D2 receptor KO mice. In contrast, motor stimulation by a D1 dopamine agonist was not attenuated in the KO mice. The A2A receptor dependence of MPEP-induced motor stimulation was investigated further using a postnatal forebrain-specific conditional (Cre/loxP system) KO of the A2A receptor. MPEP loses the ability to stimulate locomotion in conditional KO mice, suggesting that this mGlu5 antagonist effect requires the postdevelopmental action of striatal A2A receptors. The potentiation of mGlu5 antagonist-induced motor stimulation by an A2A antagonist and its dependence on both D2 and forebrain A2A receptors highlight the functional interdependence of these receptors. These data also strengthen a rationale for pursuing a combinational drug strategy for enhancing the antiparkinsonian effects of A2A and mGlu5 antagonists.