The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrenergic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for G(i)-mediated inhibition of cAMP accumulation, whereas the G(s) response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as G(i) activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of G(i) but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal G(i) and G(s) responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the alpha(2a) adrenergic receptor.