Understanding the process of Ca(2+)/Mg(2+)exchange during muscle excitation and relaxation is fundamental to elucidating the mechanism of Ca(2+)-regulated muscle contraction. During the resting phase, the C-domain of cardiac troponin C may be occupied by either Ca(2+)or Mg(2+). Here, complexes of recombinant cardiac troponin C(81-161) and the N terminus of cardiac troponin I, representing residues 33-80, were generated in the presence of saturating Mg(2+). Heteronuclear multi-dimensional nuclear magnetic resonance experiments were used to obtain backbone assignments of the Mg(2+)-loaded complex. In the presence of cardiac troponin I, the affinity of site IV for Mg(2+)is increased. Comparison of Mg(2+)and Ca(2+)-loaded complexes reveals that chemical shift differences are primarily localized to metal-binding sites III and IV, defining positions within these sites that have distinct Ca(2+)/Mg(2+)conformations. The observed transition from the Mg(2+)-loaded to Ca(2+)-loaded form demonstrates that sites III and IV fill simultaneously with Ca(2+)displacing Mg(2+). However, even in the absence of excess Ca(2+), Mg(2+)does not readily displace Ca(2+)in the isolated binary complex. Thus, the Mg(2+)-loaded conformer may only represent a small fraction of the total cardiac troponin C found in the sarcomere.