Carbon-13 nuclear magnetic resonance (¹³C-NMR) spectral analyses of the cultured fruit body of Grifola frondosa and the extracted glucan fractions were performed. NMR spectra of the fruit body were measured by suspending the powdered material in D₂O. The spectrum of the lyophilized fruit body showed signals of mono-, oligo-, and polysaccharides and fatty acids. The spectrum of the fruit body defatted with 80% ethanol showed signals attributable to poly-saccharides. By comparison with the spectra of extracts from the fruit body, these signals were assigned to α- and β-glucans. Well-resolved signals were also obtained in the spectra of residues extracted with hot water, and cold and hot alkali. These results suggest that (1) the majority of glucans presented in the fruit body has high motional freedom, (2) this method is suitable for studies of the assembly of biopolymers and the ultrastructure of fungal fruit bodies, (3) this method could be applicable for the standardization of commercially grown mushrooms.