This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an alpha-helical secondary structure with ESEEM spectroscopy using a H-2-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. H-2-SL distance distributions from the MD results indicated that H-2-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the alpha-helical M2 delta peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with H-2-labeled Leu d(10) at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an alpha-helix (3.6 residues per turn). Strong H-2 modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The H-2 modulation depth observed for H-2-labeled d(10) Leu was significantly enhanced (X4) when compared to previous ESEEM measurements that used H-2-labeled d(8) Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of H-2 modulation and the corresponding Fourier Transform (FT) peak intensity for H-2-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different H-2-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (similar to 50 mu M).