We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.