The interactive residues for mouse mAb NC10.8, which binds a superpotent guanidinium sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)guanidinoacetic acid with high affinity (Kd = 5 nM), were examined by using radioligand competitive binding, photoaffinity labeling, absorption and fluorescence spectroscopy, computer-aided molecular modeling, and site-directed mutagenesis. Competitive ligand analogue binding data revealed important structural features and a pH sensitivity for ligand binding. Spectroscopy of the sweetener-mAb complex revealed ligand-induced fluorescence quenching and the presence of a charge-transfer band. Site-directed mutagenesis of L:96W abolished the ligand-induced fluorescence quenching and reduced Ab affinity. The apparent Kd increased from 5 nM to more than 200 nM after such modification. A theoretical model of the Fv region was generated with use of a knowledge-based algorithm, and this model was used to identify the locations of key residues in the complementarity determining regions. These experimental and theoretical studies support the prediction that the sweetener ligand coordinates with the following residues: L:34H contacts the cyanophenyl ring, L:27DR forms a salt bridge with the acetic acid moiety, L:96W forms a pi-pi interaction with the cyanophenyl ring, and H:95E contacts the positively charged aryl nitrogen. These studies are important to our understanding of Ab-ligand specificity and may also shed light on the important chemical motifs responsible for elevated levels of sweetness potency in organic compounds.