A high affinity Ca(2+)-binding domain which is located in a middle portion of the large intracellular loop of the Na(+)-Ca2+ exchanger contains two highly acidic sequences, each characterized by three consecutive aspartic acid residues (Levitsky DO, Nicoll DA, and Philipson KD (1994) J Biol Chem 269: 22847-22852). This portion of the protein provides secondary Ca2+ regulation of the exchanger activity. To determine number of Ca2+ binding sites participating in formation of the high affinity domain, we isolated polypeptides of different lengths encompassing the domain and measured 45Ca2+ binding. The fusion proteins containing the high affinity domain were obtained in a Ca(2+)-bound form and as evidenced by shifts in there mobility in SDS-polyacrylamide gels after EGTA treatment. The Ca2+ binding curves obtained after equilibrium dialysis reached saturation at 1 microM free Ca2+, Kd value being approx. 0.4 microM. The Ca2+ binding occurred in a highly cooperative manner. Upon saturation, the amount of Ca2+ ion bound varied from 1.3-2.1 mol per mol protein. Proteins with an aspartate in each acidic sequence mutated lacked the positive cooperativity, had lower Ca2+ affinity and bound two to three times less Ca2+. Na(+)-Ca2+ exchangers of tissues other than heart though different from the cardiac exchanger by molecular weight most likely possess a similar Ca2+ binding site. It is concluded that, by analogy with Ca2+ binding proteins of EF-type, the high Ca(2+)-affinity domain of the Na(+)-Ca2+ exchanger is comprised of at least two binding sites interacting cooperatively.