Raman optical activity (ROA) spectroscopy is used to investigate the backbone conformation of valinomycin in methanol and dioxane solution. Experimental Raman and ROA spectral differences are interpreted by using density functional calculations, molecular dynamics, and Cartesian tensor transfer. Of the several conformers with different numbers of intramolecular hydrogen bonds which were preselected by calculations of relative energies, the dominant ones are identified on the basis of ROA. To separate the backbone signal from that of the side chains, conformational search for the isopropyl residues is performed for each backbone conformer. In dioxane, the most populated conformer does not exhibit C3 symmetry, but adopts a distorted “bracelet” structure, similar to a crystal structure. This complements previous NMR spectroscopic results that could not distinguish the nonsymmetric structures. In methanol, a different, “propeller” conformer is indicated by ROA, which has three loops resembling a standard β-turn peptide motif. Molecular dynamics simulations suggest that the propeller structure is very flexible in methanol. Spectra simulated for geometries not having the β-turn do not agree with experiment. On the basis of these results, a distinct +/− ROA couplet at ∼1335/1317 cm−1 observed in the extended amide III region is assigned to a turn in the valinomycin backbone.