Abstract Several structural features, most notably the presence of α-l-Ara f-(1 → 2)- α-d-Xyl p chains, distinguish the arabinoxyloglucans (AXGs) produced by solanaceous plants from the xyloglucans produced by other dicotyledonous plants. However, previous studies did not establish the exact order of attachment of the various side chains along the backbone of these AXGs. Therefore, oligosaccharide subunits of the AXGs secreted by suspension-cultured tobacco and tomato cells were generated by treatment of the isolated AXGs with a fungal endo- β-(1 → 4)-d-glucanase (EG). The oligosaccharides were reduced with sodium borohydride to the corresponding oligoglycosyl alditol derivatives and purified by a combination of gel-permeation chromatography, reversed-phase HPLC, and HPAE chromatography. The isolated oligoglycosyl alditols were chemically characterized by NMR spectroscopy, matrix-assisted laser-desorption/ionization time of-flight mass spectrometry (MALDITOFMS), fast-atom bombardment mass spectrometry (FABMS), FABMS/MS, and glycosyl-linkage analysis. The results confirmed that the AXGs from these species are composed of a (1 → 4)-linked β- d-Glc p backbone substituted at O-6 with various side chains. Both tobacco and tomato AXG contain α-d-Xyl p and α-l-Ara f-(1 → 2)- α-d-Xyl p side chains. However, oligosaccharide fragments of tomato AXG were also shown to contain β-d-Gal p-(1 → 2)- α-d-Xyl p and β-Ara f-(1 → 3)- α-l-Ara f-(1 → 2)- α-d-Xyl p side chains that are not present in the tobacco AXG. This is the first report of β-Ara f residues in a xyloglucan. The primary structures of 20 oligosaccharides generated by EG-treatment of tobacco AXG were determined. The generation of such a large number of oligosaccharides is due in part to the presence of O-acetyl substituents at O-6 of many of the backbone β-d-Glc p residues of tobacco AXG. The presence of either an O-acetyl or a glycosidic substituent at O-6 of β-d-Glc p residue in the AXG backbone protects the glycosidic bond of this residue from cleavage by the EG. Removal of the O-acetyl substituents prior to EG-treatment of the AXG results in oligosaccharide fragments that are smaller than those produced by EG-treatment of the O-acetylated AXG. Therefore, analysis of the complex mixture of oligosaccharides obtained by EG-treatment of native tobacco AXGs provides information regarding the distribution of AXG side chains that would be lost if the AXG is de- O-acetylated prior to EG-treatment. Furthermore, the large library of oligosaccharide fragments generated by this approach revealed additional correlations between the structural features of AXGs and diagnostic chemical shift effects in their 1H NMR spectra.