It is well known that glucagon-like peptide-1 (7-36 amide) (tGLP-1) is a potent insulinotropic hormone with powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the intracellular domains of the tGLP-1 receptor that are required for its efficient coupling to adenylyl cyclase because cAMP is the primary candidate second messenger coupling tGLP-1 to insulin secretion. Recently, we identified an amino acid within the third intracellular loop, K334, that was required for efficient coupling of tGLP-1 receptor to adenylyl cyclase. A similar mutagenesis-based strategy was employed here to examine the first and second intracellular loops and to further define sequences in the third loop required for the efficient coupling of the receptor to its second messengers. Receptor mutants were expressed in COS-7 cells and examined for tGLP-1 binding and cAMP stimulation. Three alanine substitution mutations, V327A, 1328A, and V331A, resulted in significantly lower tGLP-1-stimulated cAMP production without reductions in receptor expression. Analysis of the first and second intracellular loops revealed only one mutation contained within the first loop, R176A, where a significant reduction in cAMP activation was observed with normal receptor expression. These studies suggest that specific determinants of coupling for tGLP-1 receptor are primarily localized to the predicted junction of the fifth transmembrane helix and the third intracellular loop. We predict that V327, 1328, and V331 form part of a hydrophobic face that directly contacts the G protein.