In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.