Allosteric binding sites on adenosine -A1 and -A3 receptors represent attractive therapeutic targets for amplifying, in a spatially and temporally selective manner, the tissue protective actions of endogenous adenosine. This study has directly quantified the kinetics of agonist/G protein-coupled receptor interactions at the single-cell level, reflecting the physiological situation in which intracellular signaling proteins can exert major allosteric effects on agonist-receptor interactions. The association and dissociation rate constants at both A1 and A 3 receptors, and therefore the affinity of the fluorescent adenosine derivative ABA-X-BY630 (structure appears in J Med Chem 50:782-793, 2007), were concentration-independent. The equilibrium dissociation constants of ABA-X-BY630 at A1 and A3 receptors were approximately 50 and 10 nM, respectively, suggesting that, even in live cells, low agonist concentrations predominantly detect high-affinity receptor states. At A1 receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (koff = 1.95 ± 0.09 min-1) compared with the presence of the allosteric enhancer (2-amino-4,5-dimethyl-3-thienyl)(3- (trifluoromethyl)phenyl)-methanone (PD81,723; 10 μM; koff = 0.80 ± 0.03 min-1) and allosteric inhibitor 4-methoxy-N-(7-methyl- 3-(2-pyridinyl)-1-isoquinolinyl)benzamide (VUF5455; 1 μM; koff = 1.48 ± 0.16 min-1). In contrast, ABA-X-BY630 dissociation from A3 receptors was significantly slower in the absence (k off = 0.78 ± 0.18 min-1) than in the presence of the allosteric inhibitors VUF5455 (1 μM; koff = 3.15 ± 0.12 min-1) and PD81,723 (10 μM; koff = 2.46 ± 0.18 min-1). An allosteric mechanism of action has previously not been identified for PD81,723 at the A3 receptor or VUF5455 at the A 1 receptor. Furthermore, the marked enhancement in fluorescent agonist dissociation by VUF5455 in living cells contrasts previous observations from broken cell preparations and emphasizes the need to study the allosteric regulation of agonist binding in living cells.