Glycosynthases are engineered glycosidases which are hydrolytically inactive yet efficiently catalyse transglycosylation reactions of glycosyl fluoride donors, and are thus promising tools for the enzymatic synthesis of oligosaccharides. Two endo-glycosynthases, the E134A mutant of 1,3/1,4-bglucanase from Bacillus licheniformis and the E197A mutant of cellulase Cel7B from Humicola insolens, were used in coupled reactions for the stepwise synthesis of hexasaccharide substrates of 1,3/1,4-b-glucanases. Because the two endo-glycosynthases show different specificity, towards laminaribiosyl and cellobiosyl donors, respectively, the target hexasaccharides were prepared by condensation of the corresponding disaccharide building blocks through sequential addition of the glycosynthases in a ªone-potº process. Different strategies were used to achieve the desired transglycosylation between donor and acceptor in each step, and to prevent unwanted elongation of the first condensation product and polymerization (self-condensation) of the donor: 1) selection of disaccharide donors differing in the configuration of the hydroxyl substituent normally acting as acceptor, 2) temporary protection of the polymerizable hydroxyl group of the donor, or 3) addition of an excess of acceptor to decrease the probability that the donor can act as an acceptor. The best procedure involved the condensation of alactosyl or 4 II -O-tetrahydropyranyl-acellobiosyl fluorides with a-laminaribiosyl fluoride, catalyzed by E197A Cel7B, to give tetrasaccharide fluorides, which were then the donors for in situ condensation with methyl b-cellobioside catalyzed by E134A 1,3/1,4-b-glucanase. After isolation, the final hexasaccharides Galb4Glcb4Glcb3Glcb4Glcb4Glcb-OMe and Glcb4Glcb4Glcb3Glcb4Glcb4-Glcb-OMe were obtained in 70 ± 80 % overall yields.