The third intracellular loop (i3) plays a critical role in the coupling of many receptors to G-proteins. In muscarinic receptor subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are sufficient to direct appropriate G-protein coupling. The relative functional contributions of all amino acids within Ni3 was evaluated by constructing libraries of m5 muscarinic receptors containing random mutations in Ni3 and screening them using high throughput assays based on ligand-dependent transformation of NIH 3T3 cells. In receptors that retained a wild type phenotype, the pattern of functionally tolerated substitutions is consistent with the presence of three turns of an alpha helix extending from the transmembrane domain. All of the amino acid positions that tolerate radical substitutions face away from a conserved hydrophobic face that ends with an arginine, and helix-disrupting proline substitutions were not observed. All of the mutant receptors with significantly compromised phenotypes had amino acid substitutions in residues predicted to form the hydrophobic face. Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141-3146) are consistent with the presence of a single helical turn extending from the transmembrane domain, with an alanine that defines G-protein affinity. Functionally critical residues of Ni3 and Ci3 are predicted to be in close proximity where they form the G-protein-coupling domain.