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