Using random saturation mutagenesis, we have previously identified the amino acids K439, A440, and A441 in the C-terminus of the third intracellular loop (Ci3) of the m5 muscarinic receptor as being critical for G-protein coupling [Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141-3146]. In the present study, we have constructed a series of point mutants at each of these residues and characterized their functional phenotypes in order to define the structure/function relationships of each of these residues for G-protein coupling. Although a wide variety of substitutions were tolerated at K439, most caused significant increases in the EC50 of carbachol and decreases in the maximum response (Rmax). Only other basic residues were well tolerated (<10-fold increase in EC50, >70% of wild type). Acidic substitutions had the largest effects, reducing Rmax to under 20% of wild type. At A440, only the conservative substitution threonine was well tolerated. Substitutions by hydrophobic, polar, and basic residues caused 10-80-fold increases in EC50 values and in many cases also significantly reduced Rmax (<70% of wild type). In contrast, at A441 mutations selectively affected EC50 but not Rmax values. Previously we identified I216, Y217, T220, and R223 as the residues in the N-terminus of the i3 loop of m5 (Ni3) that are critical for G-protein coupling [Burstein, E. S., Spalding, T. S., and Brann, M. R. (1996) J. Biol. Chem. 271, 2882-2885]. To investigate whether there were additive contributions of Ni3 and Ci3 to G-protein coupling, the functional responses of two double mutants, R223E/K439E and Y217S/A441T, were evaluated. Though these mutations were tolerated individually, both double mutant receptors produced almost indetectable responses. Little or no changes in expression levels or ligand binding properties were detected, suggesting the observed effects were caused primarily by changes receptor/G-protein coupling. We conclude that K439 participates in G-protein activation through an ionic mechanism, that A440 fulfills a structural role forming part of the G-protein coupling pocket, and that A441 contributes to receptor affinity for G-proteins. We propose that the third intracellular loop forms a G-protein coupling pocket comprised of a positively charged "lip" and a hydrophobic core.