Electron spin echo envelope modulation (ESEEM) spectroscopy was used to distinguish between the local secondary structures of an alpha-helix and a 3(10)-helix. Previously, we have shown that ESEEM spectroscopy in combination with site-directed spin labeling (SDSL) and H-2-labeled amino acids (i) can probe the local secondary structure of alpha-helices, resulting in an obvious deuterium modulation pattern, where i+4 positions generally show larger H-2 ESEEM peak intensities than i+3 positions. Here, we have hypothesized that due to the unique turn periodicities of an alpha-helix (3.6 residues per turn with a pitch of 5.4 A) and a 3(10)-helix (3.1 residues per turn with a pitch of 5.8-6.0 A), the opposite deuterium modulation pattern would be observed for a 3(10)-helix. In this study, H-2-labeled d(10)-leucine (Leu) was substituted at a specific Leu residue (i) and a nitroxide spin label was positioned 2, 3, and 4 residues away (denoted i+2 to i+4) on an amphipathic model peptide, LRL8. When LRL8 is solubilized in trifluoroethanol (TFE), the peptide adopts an alpha-helical structure, and alternatively, forms a 3(10)-helical secondary structure when incorporated into liposomes. Larger H-2 ESEEM peaks in the FT frequency domain data were observed for the i+4 samples when compared to the i+3 samples for the alpha-helix whereas the opposite pattern was revealed for the 3(10)-helix. These unique patterns provide pertinent local secondary structural information to distinguish between the alpha-helical and 3(10)-helical structural motifs for the first time using this ESEEM spectroscopic approach with short data acquisition times (similar to 30 min) and small sample concentrations (similar to 100 mu M) as well as providing more site-specific secondary structural information compared to other common biophysical approaches, such as CD.