G protein-coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A₃ adenosine receptor (A₃-AR) agonist, ABEA-X-BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist-occupied A₃-AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A₃-AR, the fluorescent A₃-AR agonist was able to inhibit forskolin-stimulated [³H]cAMP production (pEC₅₀=8.57), and this was antagonized by the A₃-selective antagonist MRS1220 (pK_B=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC₅₀=7.34). Ligand binding to the A₃-AR on the membranes of single cells and subsequent increases in single cell [Ca²⁺]_i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small-membrane microdomains (0.2 µm²) revealed two agonist-occupied A₃-AR components with differing diffusion characteristics (diffusion coefficients=2.65x10^-8 and 1.19x10^-9 cm²/s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/µm², respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist-occupied A₃-receptor complexes, showing different motilities within the membrane of single living cells. Cordeaux, Y., Briddon, S. J., Alexander, S. P. H., Kellam, B., Hill, S. J. Agonist-occupied A₃ adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells.