A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca²⁺ affinity and the number of paired, negatively charged Ca²⁺ chelating residues in the N-terminal Ca²⁺-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and −x (X-axis), +y and −y (Y-axis), and +z and −z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca²⁺ affinities. The maximal ranges of the increases observed in the N-terminal Ca²⁺ affinity were ∼8−8.5-fold for site I, ∼4.5−5-fold for site II, and ∼11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca²⁺ affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca²⁺-binding sites reduced the N-terminal Ca²⁺ affinity. The increases in Ca²⁺ affinity observed were caused by an increase in the Ca²⁺ association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca²⁺ dissociation rates.