Adenosine A2A and dopamine D2 receptors have been shown previously to form heteromeric complexes and interact at the level of agonist binding, G protein coupling, and trafficking. Because dopamine D2 and D3 receptors show a high degree of sequence homology, A2A and D3 receptors may also interact in a similar manner. The present studies with confocal microscopy showed that A2A-yellow fluorescent protein (YFP) and D3-green fluorescent protein 2 (GFP2) receptors colocalize in the plasma membrane. Furthermore, fluorescence resonance energy transfer (FRET) analysis demonstrated that A2A-YFP and D3-GFP2 receptors give a positive FRET efficiency and are thereby likely to exist as heteromeric A2A/D3 receptor complexes. Saturation experiments with [3H]dopamine demonstrated that the A2A receptor agonist 4-[2-[[6-amino-9(N-ethyl-I?-d-ribofuranuronaminoamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS-21680) reduced the affinity of the high-affinity agonist binding state of the D3 receptor for [3H]dopamine. The A2A and D2A receptors seem to interact also at the level of G protein coupling, because the adenosine A2A receptor agonist CGS-21680 fully counteracted the D3 receptor-mediated inhibition of a forskolin-mediated increase in cAMP levels. Taken together, when coexpressed in the same neuron, A2A and D3 receptors seem to form A2A/D3 heteromeric receptor complexes in which A2A receptors antagonistically modulate both the affinity and the signaling of the D3 receptors. D3 receptor is one of the therapeutic targets for treatment of schizophrenia, and therefore, the A2A/D3 receptor interactions could provide an alternative antischizophrenic treatment.