Agonists bind with higher affinity to G protein-coupled heptahelical receptors than to uncoupled receptors. Recombinant A(1) and A(3) adenosine receptors couple well to G(i/o), but recombinant human A(2A) adenosine receptors (hA(2A)AR) couple poorly to G(s) and bind agonists with K(i) values in binding assays that are much higher than ED(50) values for functional responses such as coronary dilation and inhibition of neutrophil oxidative burst. In this study, we produced hA(2A)AR-G protein complexes in membranes derived from Sf9 cells quadruply infected with receptors and heterotrimeric G protein subunits. The composition of G(beta) markedly influences coupling such that A(2A)AR-alpha(s)beta(1)gamma(2) are 8 +/- 2% coupled whereas equivalently expressed A(2A)AR-alpha(s)beta(4)gamma(2) are 40 +/- 2% coupled. Hence, we were able for the first time to accurately measure high-affinity agonist binding to hA(2A)AR. The agonist 2-[2-(4-amino-3-[(125)I]iodophenyl)ethylamino]adenosine binds to coupled and uncoupled hA(2A)AR with K(D) values of 0.46 nM and 26 nM, respectively, a difference in affinity of 57-fold. The addition of GTPgammaS converts all receptors to the low-affinity state. A(2A)AR coupling does not influence binding of antagonists including, (125)I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol ((125)I-ZM241385), K(D) = 0.5 nM. Based on a comparison of high-affinity binding sites, N(6)-3-iodo-2-chlorobenzyladenosine-5'-N-methyluronamide is only 8-fold A(3) selective (A(2A Ki, H) = 18.3 +/- 3.2 nM; A(3 Ki, H) = 2.4 +/- 0.3 nM) and 2-chloro-N(6)-cyclopentyladenosine is only 33-fold A(1) selective (A(2A Ki, H) = 11.0 +/- 1.9; A(1 Ki, H) = 0.3 +/- 0.1). We conclude that recombinant hA(2A)AR can form a high-affinity receptor-G protein complex with alpha(s)beta(4)gamma(2) that is useful for determining receptor selectivity.