The 1HMR spectra of the polypeptide glucagon (29 amino acid residues) dissolved in CF3COOH, and of deuterated glucagon dissolved in CF3COOD were recorded at 220 MHz. A total of 214 observable proteon resonances were assigned to 15 spectral bands I-XV. About 65 proton resonances were assigned to spectral lines of width 5-10 cps, or to narrow spectral regions. Good agreement with earlier obtained data for insulin and its A- and B-chain S-sulfonates was obtained. Spectral effects of the slow conversion of glucagon to heptatri-fluoroacetylated glucagon were found and utilized in the assignment of the spectrum of natural glucagon. © 1969.