The structure of skeletal troponin C (sTnC) comprises two independently folded domains connected by an alpha-helical loop (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659). Both the NH2-terminal domain TR1C (9-84) and the COOH-terminal domain TR2C (89-159) can be obtained by limited tryptic cleavage. Here we report on proton and cadmium-113 NMR studies of Ca2+, Mg2+, and Cd2+ binding to these two separated domains. Our studies indicate that both halves retain a structure in the apo- and Ca2+-forms which resembles that of the intact protein. The events accompanying Ca2+ binding to these fragments are consistent with a biphasic binding pattern for sTnC, where the two sites in the COOH-terminal half are filled before those in the NH2-terminal half. Mg2+ only binds strongly to the two calcium-binding sites in the COOH-terminal half. The binding of this metal ion gives rise to a similar conformation for TR2C as that obtained with Ca2+. Whereas the binding of Ca2+ to the COOH-terminal part of sTnC takes place in a positive cooperative manner, the binding of this metal ion to TR2C occurs sequentially. Nevertheless, both Ca2+ ions bound to TR2C are in slow exchange (koff less than 10s-1). The binding of Mg24 both to sTnC and TR2C follows a sequential pattern with one site in slow exchange (koff less than 20 s-1) and one site in fast exchange (koff greater than 800 s-1). Binding of Cd2+ to TR2C occurs in a positive cooperative manner and results in the same conformation as observed with Ca2+. Cadmium-113 NMR spectra obtained at 5 degrees C confirm that two strong Cd2+-binding sites are localized in the COOH-terminal half and two weaker ones in the NH2-terminal half. It is concluded that these two proteolytic fragments comprise reasonable structural models for intact sTnC.