To test the hypothesis that G protein-coupled receptors consist of multiple autonomous folding domains, the rat m3 muscarinic acetylcholine receptor was "split" in all three intracellular (i1-i3) and all three extracellular loops (o2-o4). The six resulting polypeptide pairs (Ni1.Ci1, No2.Co2, etc.) were coexpressed in COS-7 cells and studied for their ability to bind muscarinic ligands and to activate G proteins. In addition, immunocytochemical and ELISA studies were carried out to study the expression and subcellular localization of the individual receptor fragments. Interestingly, all N- and C-terminal receptor fragments studied (except Ni1, which contained only the first transmembrane domain) were found to be localized to the plasma membrane, even when expressed alone. Coexpression of three of the six polypeptide pairs, generated by splitting the m3 muscarinic receptor in the i2, o3, or i3 loop, resulted in receptor complexes (Ni2.Ci2, No3.Co3, and Ni3.Ci3, respectively), which were able to bind muscarinic agonists and antagonists with high affinity. The No3.Co3 and Ni3.Ci3 polypeptide combinations, but not the Ni2.Ci2 complex, were also able to stimulate carbachol-dependent phosphatidyl inositol hydrolysis to a similar maximum extent as the wild type m3 muscarinic receptor. These findings strongly suggest that G protein-coupled receptors are composed of several independent folding units and may shed light on the molecular mechanisms governing receptor assembly and membrane insertion.