The effects of three mutations on the EF-hand Ca2+/Mg2+ binding site of smooth muscle myosin regulatory light chain (RLC) were studied: D5S, in which an aspartate is replaced by a serine in position 5 of the loop; D9E, in which an aspartate is replaced by a glutamate in position 9; and D12E, in which the aspartate in position 12 is replaced by a glutamate. All possible combinations of the three mutations were produced. The single mutants D5S and D9E and the double mutant D5S/D9E have low affinity for Ca2+. All the mutants containing mutation D12E are Ca2+-specific and have higher affinities than wild type, even when containing mutations D5S or D9E. All of the mutants studied have lower affinity for Mg2+ than the wild-type protein. As expected, the changes in binding free energy that each mutant produces depend on the residues present at the other positions of the site, since the mutated positions are very close in the protein structure. Coupling energies are about the same for all pairs of mutants when binding Ca2+, but can have different values when binding Mg2+. D5S and D9E have a large negative coupling energy for Mg2+ binding which suggests an interaction between these two positions. When mutation D12E is present, the coupling energy for Mg2+ binding between D5S and D9E is much lower, suggesting that this interaction occurs only if an aspartate is in position 12. Glutamate in position 9 may be able to coordinate Mg2+ directly in the double mutant D5S/D9E.