The possible molecular basis for the previously described antagonistic interactions between adenosine A ₁ receptors (A ₁ R) and dopamine D ₁ receptors (D ₁ R) in the brain have been studied in mouse fibroblast Ltk ⁻ cells cotransfected with human A ₁ R and D ₁ R cDNAs or with human A ₁ R and dopamine D ₂ receptor (long-form) (D ₂ R) cDNAs and in cortical neurons in culture. A ₁ R and D ₁ R, but not A ₁ R and D ₂ R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A ₁ R/D ₁ R heteromerization disappeared after pretreatment with the D ₁ R agonist, but not after combined pretreatment with D ₁ R and A ₁ R agonists. A high degree of A ₁ R and D ₁ R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A ₁ R and D ₂ R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A ₁ R agonist caused coclustering (coaggregation) of A ₁ R and D ₁ R, which was blocked by combined pretreatment with the D ₁ R and A ₁ R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D ₁ R and A ₁ R agonists, but not with either one alone, substantially reduced the D ₁ R agonist-induced accumulation of cAMP. The A ₁ R/D ₁ R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A ₁ R of D ₁ R receptor signaling in the brain. The persistence of A ₁ R/D ₁ R heteromerization seems to be essential for the blockade of A ₁ R agonist-induced A ₁ R/D ₁ R coclustering and for the desensitization of the D ₁ R agonist-induced cAMP accumulation seen on combined pretreatment with D ₁ R and A ₁ R agonists, which indicates a potential role of A ₁ R/D ₁ R heteromers also in desensitization mechanisms and receptor trafficking.