Abstract Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the G i/o-coupled adenosine A 1 receptor (A 1R) and G q/11-coupled P2Y 1 receptor (P2Y 1R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope-tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617–7622], although it remained to be determined whether this hetero-oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A 1R and P2Y 1R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A 1R with P2Y 1R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A 1R and P2Y 1R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero-oligomeric complexes in the rat brain.