Previously, an electron spin echo envelope modulation (ESEEM) spectroscopic approach was established to probe the local secondary structure of membrane proteins and peptides utilizing site-directed spin-labeling (SDSL). In this method, the side chain of one amino acid residue is selectively H-2-labeled and a nitroxide spin label is strategically placed 1, 2, 3, or 4 amino acids away from the H-2-labeled amino acid (denoted as i +/- 1 to i +/- 4, i represents the H-2 labeled amino acid). ESEEM can detect the dipolar coupling between the nitroxide spin label and H-2 atoms on the amino acid side chain. Due to the periodicity of different secondary structures, different ESEEM patterns can be revealed to probe the structure. For an alpha-helical structural component, a H-2 ESEEM signal can be detected for i +/- 3 and i +/- 4 samples, but not for i +/- 1 or i +/- 2 samples. Several H-2-labeled hydrophobic amino acids have been demonstrated in model system that can be utilized to identify local secondary structures via this ESEEM approach in an extremely efficient fashion. In this study, the ESEEM approach was used to investigate the rod 2B region of the full-length intermediate filament protein human vimentin. Consistent with previous EPR and X-ray crystallography results, our ESEEM results indicated helical structural components within this region. Thus, this ESEEM approach is able to identify alpha-helical structural components despite the coiled-coil nature of the vimentin structure. The data show that the human vimentin rod 2B adapted a typical alpha-helical structure around residue Leu309. This result is consistent with the X-ray data from fragmented protein segments and continuous wave EPR data on the full-length vimentin. Finally, the ESEEM data suggested that a local secondary structure slightly different from a typical alpha-helix was adopted around residue 340.