Complete assignments of nitrogen-15 resonances of gramicidin S have been made in dimethyl sulfoxide, trifluoroethanol, and in a solvent mixture of dimethyl sulfoxide (50%) and methanol (50%). The assignments are achieved by utilizing the secondary structure of gramicidin S, by comparing the nitrogen-15 spectrum of gramicidin S with that of di-N-methylphenylalanine-gramicidin S and by taking into account the distinguishable value of nitrogen-15 chemical shift for valine in model compounds. Deuterium substitution for labile peptide protons was performed to delineate solvent shielded and deshielded peptide nitrogens and to substantiate further the signal assignments. The solvent titration on going from dimethyl sulfoxide to trifluoroethanol was also performed and shown to have a large deshielding effect on the peptide nitrogen whose corresponding peptide carbonyl, within the peptide moiety, was accessible to the trifluoroethanol solvent. © 1978, American Chemical Society. All rights reserved.