The EF-hand motif (helix–loop–helix) is a Ca2+-binding domain that is common among many intracellular Ca2+-binding proteins. We applied Fourier-transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17-residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D-type amino acid, were investigated to disturb the α-helical conformation of helix F systematically. These D-type-substituted peptides showed no band at about 1555 cm−1 even in the Ca2+-loaded state although the native peptide (L-type only) showed a band at about 1555 cm−1 in the Ca2+-loaded state, which is assigned to the side-chain COO− group of Glu at the 12th position, serving as the ligand for Ca2+ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca2+ and the side-chain COO− group of Glu at the 12th position. Implications of the COO− antisymmetric stretch and the amide-I′ of the synthetic peptide analogues of the Ca2+-binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.