Biological functions for a large class of calmodulin‐related proteins, such as target protein activation and Ca²⁺ buffering, are based on fine‐tuned binding and release of Ca²⁺ ions by pairs of coupled EF‐hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK_a values for all aspartic and glutamic acid residues in apo calbindin D_9k, a representative of calmodulin‐related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca²⁺ ligands with high pK_a under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca²⁺ ligand of each site, and that the potential is significantly more negative at the N‐terminal binding site. Proteins 2001;45:129–135. © 2001 Wiley‐Liss, Inc.