We have investigated potential Gi and Gs coupling domains within the intracellular regions of the alpha2AAR subtype using a series of nine chimeric mutations. The second intracellular loop (ICL2, amino acids 133-149) and the amino- and carboxyl-terminal regions of the third intracellular loop (ICL3, amino acids 218-235 and 355-371, respectively) of the cloned human alpha2AAR were substituted with the analogous sequence from either the Gs-coupled beta2AR or the Gi-coupled serotonin type 1A receptor (5-HT1AR). Mutant and wild type alpha2AAR were stably expressed in Chinese hamster ovary cells and functional coupling of each receptor to Gi and Gs was assessed in membrane adenylyl cyclase assays. Substitution of 5-HT1AR sequence into ICL2 ablated coupling to Gs but not to Gi, whereas substitution of beta2AR sequence significantly depressed coupling to Gi but not to Gs. Thus, the ICL2 of the alpha2AAR contains elements essential for both signaling pathways. Substitution of either the amino- or carboxyl-terminal segments of ICL3 with 5-HT1AR sequence ablated agonist stimulation of adenylyl cyclase activity (without affecting inhibition), suggesting that both domains are necessary for alpha2AAR coupling to Gs. In contrast, individual substitution of beta2AR sequence into ICL3 amino or carboxyl termini had no appreciable effect on Gi coupling. Concomitant substitution of beta2AR sequence into both regions substantially impaired Gi coupling, implying that each is capable of independently supporting functional coupling. Substitution of 5-HT1AR at either locus had no effect on Gi coupling. Thus, for Gs coupling, these two domains within ICL3 are both required for functional coupling. However, for Gi coupling, the alpha2AAR appears to have two distinct regions within ICL3 that are capable of supporting Gi coupling independently. There has been no previous elucidation of a receptor having redundant, fully competent domains for coupling to a single class of G-protein. Such duplicity of functional domains within alpha2AR may suggest strong evolutionary pressure to maintain Gi coupling.