Stable cell lines that express the canine-derived A2a adenosine receptor (A2aAR) have been generated. Using a previously characterized anti-A2aAR antibody probe, we have identified the recombinant receptor protein and examined the desensitization process of this G protein-coupled receptor. Agonist exposure induced a rapid desensitization of A2aAR-stimulated adenylyl cyclase activity. This was associated with reduced affinity of the receptor for the A2aAR-selective agonist [3H]CGS21680 and agonist-stimulated phosphorylation of the receptor protein. Agonist-stimulated A2aAR sequestration into a light membrane fraction was also detected over the same time frame but, whereas inhibition of this process did not affect the extent of desensitization, the rapid recovery normally observed after short term agonist exposure was dramatically reduced. Long term agonist treatment resulted in the down-regulation of A2aARs and up-regulation of Gi alpha 2 and Gi alpha 3, as determined by immunoblotting. Recovery of A2aAR function after agonist removal required several hours and was associated with the return of receptor levels to control values. In contrast, inactivation of Gi proteins by pertussis toxin treatment did not alter the extent of agonist-induced desensitization observed. Neither short nor long term desensitization could be mimicked by elevation of intracellular cAMP levels alone. Therefore, these data suggest that A2aAR desensitization is mediated by multiple, temporally distinct, agonist-dependent processes. Agonist-stimulated phosphorylation of the receptor may induce short term desensitization by impairing receptor-Gs coupling, whereas long term down-regulation of receptor number and up-regulation of inhibitory G proteins mediate long term adaptation.