The secondary structure of the N-extension of cardiac troponin I (cTnI) was determined by measuring the distance distribution between spin labels attached to the i and i + 4 residues: 15/19, 23/27, 27/31, 35/39, and 43/47. All of the EPR spectra of these regions in the monomeric state were broadened and had a amplitude that was reduced by two-thirds of that of the single spin-labeled spectra and was fit by two residual distance distributions, with a major distribution one spreading over the range from 1 to 2.5 nm and the other minor peak at 0.9 nm. Only slight or no obvious changes were observed when the extension was bound to cTnC in the cTnI-cTnC complex at 0.2 M KCl. However, at 0.1 M KCl, residues 43/47, located at the PKC phosphorylation sites Ser42/44 on the boundary of the extension, exclusively exhibited a 0.9 nm peak, as expected from α-helix in the crystal structure, in the complex. Furthermore, 23/27, which is located on the PKA phosphorylation sites Ser23/24, showed that the major distribution was markedly narrowed, centered at 1.4 nm and 0.5 nm wide, accompanying the spin label immobilization of residue 27. Residues 35 and 69 at site 1 and 2 of cTnC exhibited partial immobilization of the attached spin labels upon complex formation. The results show that the extension exhibited a primarily partially folded or unfolded structure equilibrated with a transiently formed α-helix-like short structure over the length. We hypothesize that the structure binds at least near sites 1 and 2 of cTnC and that the specific secondary structure of the extension on cTnC becomes uncovered when decreasing the ionic strength demonstrating that only the phosphorylation regions of cTnI interact stereospecifically with cTnC.