Activation of the thin filament in striated muscles requires both the binding of Ca2+ to the N-domain of troponin C (TnC) and the binding of myosin crossbridges to actin, which has been shown to alter the C-domain conformation. Here we have evaluated the structural and functional consequences of divalent cation exchange in skeletal and cardiac TnC (sTnC and cTnC). We have used intrinsic tyrosine fluorescence, circular dichrosim (CD), and the fluorescent nonspecific hydrophobic probe bis-ANS to monitor changes in domain conformation in response to Ca2+ and Mg2+ binding in the sTnC, cTnC, and in a cTnC mutant in which the invariant Glu residue at the 12th position of the calcium binding loops III and IV were substituted with Asp (cTnCDD). Ca2+ binding causes an increase in Tyr fluorescence and α-helical content in sTnC and cTnC, but not in cTnCDD. Ca2+ induced C-domain opening characteristic of sTnC and cTnC was also greatly reduced in cTnCDD, as measured by bis-ANS fluorescence. Thus the Asp to Glu substitutions appear to prevent the C-domain from opening. Bis-ANS Ca2+ titrations also showed that high Ca2+ concentrations may be sufficient to open the N-domain of cTnC, which was reported to remain in the closed conformation in the Ca2+-bound state. Lastly, bis-ANS Mg2+ titrations indicate that Mg2+ does not cause domain opening in either cTnC or cTnCDD. The closed conformation of the Mg2+-bound C-domain of TnC implies a different mechanism of interaction with TnI than that in the presence of Ca2+ and suggests that the Mg2+- Ca2+exchange in TnC may contribute to the thin filament activation of muscle contraction. This conclusion is consistent with our observation that physiological concentrations of Mg2+ significantly lower the Ca2+-sensitivity of reconstituted cardiac thin filaments.