To deepen our understanding of Ca2+-activation in calmodulin-dependent reactions, the properties of Ca binding to calmodulin were examined by dual-wavelength spectrophotometry using a Ca indicator, tetramethylmurexide. The Scatchard plot for Ca binding to calmodulin was linear, indicating the occurrence of homogeneous independent binding sites. The apparent binding constant of calmodulin for Ca2+ (K) was (1.96 +/- 0.05) X 10(5) M-1 (20), and the number of binding sites (n) was 3.36 +/- 0.04 (20) mol/mol in reaction medium containing 100 mM KCl, 20 mM MOPS-KOH (pH 6.80), and 0.12 mM tetramethylmurexide at 20 degrees C. K was strongly dependent on the concentration of KCl, while n was independent of it. On the assumption that this is due to the effect of ionic strength (I), K may be described as follows: log K = 6.73-3.2 [2 square root I/(1 + square root I)-0.4 X I] (0.006 less than or equal to I less than or equal to 0.256, pH 6.80, at 20 degrees C). Mg2+ decreased K, which could be expressed as K/(1 + 130 [Mg] ) ( [Mg] in mol (M) ) at the ionic strength of 0.106, pH 6.80 at 20 degrees C. n was also decreased in the presence of Mg2+. This suggests that Mg2+ may have effects other than simple competition. The pH of the medium affected K or n very little at around neutral pH. This may correspond to the fact that calmodulin is an acidic protein with pI congruent to 4.3. K or n was only slightly dependent on the temperature. This corresponds to the calorimetric result that the enthalpy change on Ca binding was very slight and endothermic. Based on these findings, much of the discrepancy among the results already reported on Ca-binding can be explained. Activation of a calmodulin-dependent reaction is discussed on the basis of Ca binding to calmodulin under physiological conditions.