The effect of Mg2+ on Ca2+ bound to glycerinated rabbit psoas muscle was studied by means of a double-isotope technique. The troponin-C content of the fibers was analyzed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and found to be 0.5 mumol/g fiber protein. In the absence of Mg2+ the fibers bound a maximum of 3.5 mumol Ca2+/g protein. This value could be readily accounted for in terms of the four Ca2+-binding sites of troponin-C and the two divalent cation-binding sites of myosin. In the presence of 1 mM Mg2+ the entire Ca2+ titration curve was shifted downward with a maximum bound Ca2+ of slightly more than 2 mumol/g, or 4 mol Ca2+/mol troponin-C. Further increase in Mg2+ concentration to 10 mM had little effect on Ca2+ binding when the free Ca2+ concentration was in the upper part of the physiological range (5 . 10(-7)-5 . 10(-6) M) but caused a marked reduction when the Ca2+ concentration was low. The results are consistent with biochemical data showing two groups of (Mg2+/Ca2+)-binding sites. One group, located in myosin, has a high affinity for Mg2+ while the other group, located on troponin-C, has a low affinity for Mg2+. If the free Mg2+ concentration in muscle is in the range of 2--5 mM, as suggested by recent data, it can be inferred that the binding sites on myosin will never be occupied by Ca2+ under physiological conditions.