In this work we describe calcium binding to two synthetic 34-residue peptides, determined by 1H-nmr spectroscopy. The peptides investigated, SCIII and SCIV, encompass the calcium-binding sites III and IV, respectively, of troponin-C. In the absence of calcium it has previously been shown that each of these peptides possesses little regular secondary structure. Further, the 1H-nmr spectra of an equimolar mixture of both of these apo-peptides (apo-SCIII/SCIV) shows that little interaction occurs between peptides. Upon calcium binding the spectral changes that occur to SCIII/SCIV are consistent with global conformational changes in both peptides. We have shown previously that these conformational changes are a product of calcium binding to SCIII and SCIV to form a two-site heterodimer Ca2-SCIII/SCIV. It is proposed that this calcium-induced folding proceeds via calcium binding to SCIII to form Ca-SCIII, peptide association with apo-SCIV to form the heterodimer Ca-SCIII/SCIV, and calcium binding to form Ca2-SCIII/SCIV. The dissociation constants involved in this pathway, K1, Kd, and K2, respectively, have been determined by stoichiometric calcium titration of SCIII/SCIV, monitored by 1H-nmr spectroscopy. Using this procedure it has been determined that K1 = 3 microM, Kd = 10 microM, and K2 = 2 microM.