Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1→4)-β-D-glucasane (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Gle4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosylxylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Gle) residues numbered -3, -2, + 2 and + 3may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. Tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide- specific α-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXG, XLXG XXLG and XLLG in native tamarind and nasturtium seed xylogucans with those in NXET digest of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose substitution at glucose residues -4, -2, + 1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain- cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.