In an attempt to delineate the structural requirements necessary for agonist-induced desensitization, we have stably expressed wild-type and mutant A2a adenosine receptors (A2aARs) in Chinese hamster ovary cells and examined the effects of agonist pretreatment on adenylyl cyclase activity in subsequently isolated membranes. Deletion of 95 amino acids from the carboxyl-terminus of the A2aAR, thereby removing 10 potential phosphorylation sites, failed to alter radioligand binding, adenylyl cyclase activation, or functional desensitization parameters as compared with the wild-type receptor. However, simultaneous mutation of Thr 298 and Ser 305 to Ala residues attenuated the desensitization observed in response to short-term (30 min) agonist treatment while not blocking the ability to desensitize after long-term (24 h) agonist exposure. Individual mutation of these residues revealed that mutation of Thr 298 alone was sufficient to diminish both short-term desensitization and agonist-stimulated receptor phosphorylation. These data suggest that while the majority of the A2aAR carboxyl-terminus is dispensable with regard to observing functional desensitization, the integrity of Thr 298 is essential for observing agonist-stimulated receptor phosphorylation and short-term desensitization but not long-term desensitization of A2aAR function.