Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing H-2 isotopic labeling and site-directed spin labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was H-2-labeled, such as H-2-labeled d(10)-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with H-2-labeled d(10)-Leu, four Leu residues within the AChR M2 delta peptide were fully mapped out using this ESEEM method. Unique H-2-ESEEM patterns were observed with the H-2-labeled d(10)-Leu for the AChR M2 delta alpha-helical model peptide. For proteins and peptides with an alpha-helical secondary structure, deuterium modulation can be clearly observed for i +/- 3 and i +/- 4 samples, but not for i +/- 2 samples. Also, a deuterium peak centered at the H-2 Larmor frequency of each i 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an alpha-helix from a pi-helix or 3(10)-helix. Moreover, H-2 modulation depth for ESEEM samples on Leul0 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2 delta peptide as suggested by previous MD simulations and NMR experiments.