A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A 1 or A 3 adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A 3-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A 1 and A 3 receptors were 1.45 ? 0.05 and 0.57 ? 0.07 min +, respectively. At the A3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5 -(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A 3-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A 1 receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC 50 values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A 3-receptor dimer yielded values of 6.0 ? 0.1, 5.9 ? 0.1, and 5.2 ? 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.