Functional interactions between adenosine A2A and dopamine D2 receptors have been demonstrated both at the D2 agonist-binding and second messenger levels. The present studies use a [3H]dopamine-binding assay as a sensitive measure of A2A receptor-mediated modulation of D2 receptors. Co-incubation with an A2A receptor agonist increased the Kd value of high-affinity [3H]dopamine-binding sites of the D2 receptor without changing their Bmax values in a cotransfected cell line. This interaction was shown to be subtype specific, as the A2A receptor agonist did not modulate the affinity of the D1 receptor for [3H]dopamine. The domains of the D2 receptor important for the A2A/D2 receptor interaction were studied with chimeric dopamine D2/D1 receptors. The results showed that the A2A receptor agonist still strongly reduced the affinity of a D2/D1 chimera with the sixth transmembrane (TM) domain and third extracellular loop from the D1 receptor. However, the A2A receptor agonist was not able to modulate a D2/D1 chimeric receptor containing the fifth and sixth TM domains and the third intracellular and extracellular loops from the D1 receptor, indicating that the fifth TM domain and/or the third intracellular loop may be involved in the interaction between A2A and D2 receptors.