Dextransucrase from Streptococcus sanguis 10558 was found to synthesize alpha-(1-->6), alpha-(1-->3), and alpha-(1-->2) linkages during an acceptor-dependent glucosyl transfer reaction. Normally, new glucosyl residues are added at C-6 of monosaccharide acceptors. However, sugars blocked at C-6 also can serve as good acceptors. The disaccharide and trisaccharide products formed when methyl 6-bromo-6-deoxy-alpha-D-glucopyranoside was used as acceptor were isolated and characterized. Both were found to contain only alpha-(1-->3) glycosidic bonds. This supports the hypothesis that when C-6 is blocked the acceptor binds to the enzyme in a flipped orientation, resulting in an approximate exchange in space of the C-3 and C-6, thereby putting C-3 adjacent to the active site. The second alpha-(1-->3) links in the trisaccharide are formed by a single-chain mechanism without release of the intermediate disaccharide. With maltose as acceptor, new glucosyl residues are added at C-6'. However, if that position is blocked with a bromine atom, the resulting compound, 6'-bromo-6'-deoxy-maltose, can still serve as an acceptor. The product in this case was isolated and characterized. The new glycosidic link was found to be alpha-(1-->2).