NMR spectral analysis of the novel amino acid, diphthamide, in elongation factor 2 which is ADP-ribosylated by diphtheria toxin suggests that it is 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. Ribosyl-diphthamide was prepared by enzymatic hydrolysis of ADP-ribosyl-elongation factor 2 and three compounds were produced by its chemical hydrolysis (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10717-10720). Proton NMR spectroscopy in 2H2O of diphthine demonstrated the elements of histidine minus the carbon 2 proton plus 14 additional nonexchangeable protons. These protons were attributed to an extensive modification at carbon 2 of the imidazole ring. Proton NMR spectroscopy in 2H2O of ribosyl-diphthamide showed only those protons seen in diphthine plus those expected of a ribofuranosylmoiety. Chemical shift dependence on pH was demonstrated for the histidine-derived protons as well as several protons of the modifying side chain. Natural abundance carbon 13 NMR spectroscopy of ribosyldiphthamide also showed the elements of histidine and ribose plus 7 additional carbon atoms attributed to the modification of the imidazole ring. Based on the NMR spectral properties of the anomeric proton of ribosyldiphthamide we propose that the ribose is linked to one of the nitrogens of the histidine imidazole ring.