Bitter taste is one of the five basic taste modalities, and is mediated by 25 bitter taste receptors (T2Rs) in humans. How these few receptors recognize a wide range of structurally diverse bitter compounds is not known. To address this question, structural and functional studies on T2Rs are necessary. Quinine is a natural alkaloid and one of the most intense bitter tasting compounds. Previously it was shown that quinine activates T2R4, however, whether T2R4 has only one binding site for quinine, and the amino acids on the receptor involved in binding to quinine remain to be determined. In this study, the ligand binding pocket on T2R4 for quinine was characterized using a combination of approaches. These included molecular model guided site-directed mutagenesis, characterization of the expression of the mutants by flow cytometry, and functional characterization by cell based calcium imaging. Twelve mutations were made in T2R4 and their expression and function were characterized. Results show that the ligand binding pocket of T2R4 for quinine is situated on the extracellular side, and is formed by the residues present on the transmembrane regions TM3, TM4, and extracellular loop regions ECL2 and TM6-ECL3-TM7 interface. Further, this study identified the following amino acids : A90, F91, Y155 N173, T174, Y258 and K270 to play an important role in quinine binding to T2R4. The detailed study of residues interacting with ligand will help in understanding how various ligands interact with T2Rs, and facilitate the pharmacological characterization of potent antagonists or bitter taste blockers. The characterization of novel ligands, including bitter taste blockers will help in dissecting the signaling mechanism(s) of T2Rs, and help in the development of novel therapeutic tools for the food and drug industry.