The ligand-induced activation of G protein-coupled receptors (GPCRs) is predicted to involve pronounced conformational changes on the intracellular surface or the receptor proteins. A reorientation of the cytoplasmic end of transmembrane domain VI (TM VI) is thought to play a key role in GPCR activation and productive receptor/G protein coupling. Disulfide cross-linking studies with solubilized, Cys-substituted mutant versions of bovine rhodopsin and the M3 muscarinic acetylcholine receptor suggested that the cytoplasmic end of TM VI is conformationally highly flexible, even in the absence of activating ligands (Farrens, D. L., et al. (1996) Science 274, 768-770; Zeng, F. Y., et al. (1999) J. Biol. Chem. 274, 16629-16640). To test the hypothesis that the promiscuous disulfide cross-linking pattern observed in these studies was caused by the use of solubilized receptor proteins endowed with increased conformational flexibility, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bonds in Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. Specifically, we used membranes prepared from transfected COS-7 cells to analyze a series of double Cys mutant M3 receptors containing one Cys residue within the sequence K484(6.29) to S493(6.38) at the cytoplasmic end of TM VI and a second Cys residue at the cytoplasmic end of TM III (I169C(3.54)). This analysis revealed a disulfide cross-linking pattern that was strikingly more restricted than that observed previously with solubilized receptor proteins, both in the absence and in the presence of the muscarinic agonist, carbachol. Carbachol stimulated the formation of disulfide bonds in only two of the 10 analyzed mutant muscarinic receptors, I169C(3.54)/K484C(6.29) and I169C(3.54)/A488C(6.33), consistent with an agonist-induced rotation of the cytoplasmic end of TM VI. These findings underline the usefulness of analyzing the structural and dynamic properties of GPCRs in their native lipid environment.