We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue Nα-acetyl[19FPhe106]TnI(104–115)amide. Dissociation constants of Kd = (3.7 ± 3.1) × 10−5 M for the apo interaction and Kd = (4.8 ± 1.8) × 10−5 M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM ≤ Kd1 ≤ 0.071 mM and 0.5 mM ≤ Kd2 ≤ 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.Key words: tropinin C, tropinin I, calcium binding, NMR studies, muscle proteins.