In a large number of intracellular regulatory proteins of the calmodulin superfamily a pair of closely interacting helix-loop-helix Ca2+ binding sites ('EF hands') constitute the functional unit--an arrangement that enables cooperative binding. We have recently made detailed experimental studies of the binding of Ca2+ ions to calmodulin, its tryptic fragments TR1C and TR2C (which each constitute a globular domain of a pair of EF hands) and calbindin D9k. Macroscopic Ca2+ binding constants have been obtained over a range of ionic strengths (0 to 0.15 M KCl). For calmodulin the measurements indicate that the two separate globular domains TR1C and TR2C retain the Ca2+ binding properties they have in the intact molecule, with positive cooperativity within each domain. The absolute value of the free energy of interaction between the two sites in each domain, a measure of the cooperativity, increases with ionic strength and is greater than or equal to 10 kJ mol-1 at 0.15 M KCl. Two-dimensional 1H NMR studies show that the addition of KCl does not alter the conformation of the protein. In the case of calbindin D9k several categories of mutants have been studied. One group encompasses the effect of protein surface charges 5 to 15 A from the Ca2+ binding sites. Two-dimensional 1H NMR shows that neither the addition of KCl, nor mutations that neutralize the surface charges, change the protein conformation. Although the global structure of calbindin D9k is largely unchanged upon binding of calcium, the structure with only one cation bound is more similar to the (Ca2+)2 form. Interestingly, the dynamical properties of the Ca(2+)-free and the (Ca2+)2-forms of calbindin differ greatly. For example, the rate of NH/ND exchange of the Ca(2+)-free form is on average 200 times faster than that of the (Ca2+)2-form. The results obtained so far point to a non-negligible entropic contribution to the observed cooperativity of Ca2+ binding.