Ca(2+)-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca(2+) concentration ([Ca(2+)](o)) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca(2+) in regulating CaSRs is the lack of adequate information about their Ca(2+)-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca(2+)-binding affinities. In this paper, we have reported the identification of three potential Ca(2+)-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca(2+)](o), similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca(2+)-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca(2+)-binding properties of the predicted sequences, we engineered two predicted continuous Ca(2+)-binding sequences individually into a scaffold protein provided by a non-Ca(2+)-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb(3+) fluorescence energy transfer. Removing the predicted Ca(2+)-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca(2+)-binding residues were shown to be involved in Ca(2+)/Ln(3+) binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca(2+)-binding sites within the extracellular domain of the CaSR.