Glial cell line-derived neurotrophic factor (GDNF) affords neuroprotection in Parkinson's disease in accordance with its ability to bolster nigrostriatal innervation. We previously found that GDNF facilitates dopamine release in a manner dependent on adenosine A(2A) receptor activation. As motor dysfunction also involves modifications of striatal glutamatergic innervation, we now tested if GDNF and its receptor system, Ret (rearranged during transfection) and GDNF family receptor alpha1 controlled the cortico-striatal glutamatergic pathway in an A(2A) receptor-dependent manner. GDNF (10 ng/mL) enhanced (by approximately 13%) glutamate release from rat striatal nerve endings, an effect potentiated (up to approximately 30%) by the A(2A) receptor agonist CGS 21680 (10 nM) and prevented by the A(2A) receptor antagonist, SCH 58261 (50 nM). Triple immunocytochemical studies revealed that Ret and GDNF family receptor alpha1 were located in 50% of rat striatal glutamatergic terminals (immunopositive for vesicular glutamate transporters-1/2), where they were found to be co-located with A(2A) receptors. Activation of the glutamatergic system upon in vivo electrical stimulation of the rat cortico-striatal input induced striatal Ret phosphorylation that was prevented by pre-treatment with the A(2A) receptor antagonist, MSX-3 (3 mg/kg). The results provide the first functional and morphological evidence that GDNF controls cortico-striatal glutamatergic pathways in a manner largely dependent on the co-activation of adenosine A(2A) receptors.