Tri-O-methylcellulose was prepared from partially O-methylated cellulose and its chemical shifts (H-1 and C-13), and proton coupling constants were assigned using the following NMR methods: (1) One-dimensional H-1 and C-13 spectra of the title compound were used to assign functional groups and to compare with literature data; (2) double quantum filtered proton-proton correlation spectroscopy (H-1, H-1 DQF-COSY) was used to assign the chemical shifts of the network of 7 protons in the anhydroglucose portion of the repeat unit; (3) the heteronuclear single-quantum coherence (HSQC) spectrum was used to establish connectivities between the bonded protons and carbons; (4) the heteronuclear multiple-bond correlation (HMBC) spectrum was used to connect the hydrogens of the methyl ethers to their respective sugar carbons; (5) the combination of HSQC and HMBC spectra was used to assign the C-13 shifts Of the methyl ethers; (6) all spectra were used in combination to verify the assigned chemical shifts; (7) first-order proton coupling constants data (J(H,H) in Hz) were obtained from the resolution-enhanced proton spectra. The NMR spectra of tri-O-methylcellulose and other cellulose ethers do not resemble the spectra of similarly substituted cellobioses. Although the H-1 and C-13 shifts and coupling constants of 2,3,6-tri-O-methylcellulose closely resemble those of methyl tetra-O-methyl-beta-D-glucoside, there are differences with regard to the chemical shifts and the order of appearances of the resonating nuclei of the methyl ether appendages and the proton at position 4 in the pyranose ring. H4 in tri-O-methylcellulose is deshielded by the acetal system comprising the beta-1-->4 linkage, and it resonates downfield. H4 in the permethylated glucoside is not as deshielded by the equitorial O-methyl group at C4, and it resonates upfield. The order of appearance of the H-1 and C-13 resonances in the spectra of the tri-O-methylcellulose repeat-unit (from upfield to downfield) are H2 < H3 < H5 < H6a < H3a < H2a < pro R H6B < H4 < pro S H6A << H1 and C6a < C3a < C2a < C6 < C5 < C4 < C2 < C3 << C1, respectively. Close examination of the pyranose ring coupling constants of the repeat unit in tri-O-methylcellulose supports the C-4(1) arrangement of the glucopyranose ring. Examination of the proton coupling constants about the C5-C6 bond (J(5,6A) and J(5,6B)) in the nuclear Overhauser effect difference spectra revealed that the C6 O-methyl group is predominantly in the gauche gauche conformation about the C5-C6 bond for the polymer in solution.