A small change in the extracellular Ca(2+) concentration ([Ca(2+)](o)) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca(2+)-sensing receptors (CaSR). Mainly through binding of Ca(2+) to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca(2+) responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca(2+)-binding sites by grafting CaSR-derived, Ca(2+)-binding peptides to a scaffold protein, CD2, that does not bind Ca(2+). In this paper, we predict more potential noncontinuous Ca(2+)-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains two to three predicted Ca(2+)-binding sites. This approach enables us to further understand the mechanisms underlying the binding of multiple metal ions to extended polypeptides derived from a location within the ECD of the CaSR, which would be anticipated to more closely mimic the structure of the native CaSR ECD. Tb(3+) luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca(2+)-dependent conformational changes in these subdomains. Removing the predicted Ca(2+)-binding ligands in site 1 and site 3 abolishes the first binding step and second binding step, respectively. Studies on these subdomains suggest the existence of multiple metal-binding sites and metal-induced conformational changes that might be responsible for the switching on and off the CaSR by the transition between its open inactive form and closed active form.