Solid-state 13C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-13C]Gly3-[2-13C]Ala4, [1-13C]Gly3-[2-13C]Leu6, [1-13C]Leu13-[2-13C]Ala15, [2-13C]Leu13-[1-13C]Ala15, and [1-13C]Leu13-[2-13C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-13C]Gly3-[2-13C]Ala4 and [1-13C]Gly3-[2-13C]Leu6 were consistent with α-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-13C]Leu13-[2-13C]Ala15, [2-13C]Leu13-[1-13C]Ala15, and [1-13C]Leu13-[2-13C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139°–145°) than in lyophilized powder (121°–139°) or crystals (129°). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (∼160°) (R. Smith, F. Separovic, T. J. Milne, A. Whittaker, F. M. Bennett, B. A. Cornell, and A. Makriyannis, 1994, J. Mol. Biol. 241:456–466). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.