G protein-coupled receptors constitute the largest family of trans membrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed-by receptor and G protein. We therefore have characterized the oligomeric status of eGFP tagged M-2 muscarinic receptor (M2R) and G(i1) by single-particle photobleaching of immobilized complexes. The method was calibrated With multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-G(i1) were indicative of a hexamer and unaffected by GTP gamma S. A complex of M2R and G(i1) was tetrameric in both, and activation by a full agonist plus GTP gamma S reduced the oligomeric size of G(i1) without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Ga-i1 by the receptor-mimic mastoparan plus GTP gamma S, and constitutively active eGFP-Ga-i1 was predominantly dimeric. The oligomeric nature of G(i1) in live CHO cells was demonstrated by means of Forster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled G alpha(i1); stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-G(i1) is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of G(i1). The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.