The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.