A(1) adenosine receptors (A(1)Rs) and adenosine deaminase (ADA; EC 3.5.4.4) interact on the cell surface of DDT1MF-2 smooth muscle cells. The interaction facilitates ligand binding and signaling via A(1)R, but it is not known whether it has a role in homologous desensitization of A(1)Rs. Here we show that chronic exposure of DDT1MF-2 cells to the A(1)R agonist, N-6-(R)-(phenylisopropyl)adenosine (R-PIA), caused a rapid aggregation or clustering of A(1) receptor molecules on the cell membrane, which was enhanced by pretreatment with ADA Colocalization between A(1)R and ADA occurred in the R-PLA-induced clusters. Interestingly, colocalization between A(1)R and ADA also occurred in intracellular vesicles after internalization of both protein molecules in response to R-PIA. Agonist-induced aggregation of A(1)Rs was mediated by phosphorylation of A(1)Rs, which was enhanced and accelerated in the presence of ADA Ligand-induced second-messenger desensitization of A(1)Rs was also accelerated in the presence of exogenous ADA, and it correlated well with receptor phosphorylation, However, although phosphorylation of A(1)R returned to its basal state within minutes, desensitization continued for hours. The loss of cell-surface binding sites (sequestration) induced by the agonist was time-dependent (t(1/2) = 10 +/- 1 h) and was accelerated by ADA. All of these results strongly suggest that ADA plays a key role in the regulation of A(1)Rs by accelerating ligand-induced desensitization and internalization and provide evidence that the two cell surface proteins internalize via the same endocytic pathway.