Metabotropic Glutamate Receptor subtype 1 alpha (mGluR1α) mediates the accumulation of IP3 and DAG, release of intracellular Ca2+ from ER, and activation of PKC and PKA, by interacting with extracellular glutamate and Ca2+. While more than 12 X-ray Structures of different forms of the extracellular domain (ECD) has been determined, the detailed binding locations of Ca2+ still remain unidentified. In this study, we first report our prediction of several Ca2+ binding sites in the ECD of mGluR1a using our newly developed computational algorithms. Putative residues involved in the calcium binding were verified using a grafting approach. Their capability to bind Ca2+ and its trivalent analog, Tb3+ were determined using fluorescence energy transfer and NMR. The substitution of charged or polar ligand binding residues with Ile at Site 1 resulted in up to 99-fold decrease in the Tb3+ binding affinity. In transiently-transfected HEK cells, mutations on these proposed ligand binding residues in the predicted calcium binding site either resulted in increase or decrease of intracellular Ca2+ response toward changes in [Ca2+]o. When treated mGluR1α with glutamate and Ca2+ together, the intracellular Ca2+ response is significantly greater than individual effects by glutamate or Ca2+, suggesting a cooperative effects by calcium binding and glutamate.