Enzymatic synthesis of 3-O-methyl-4-O-beta -D-galactopyranosyl-D-glucose (3-O-methyl-lactose) has been attempted using both galactosyltransferase and galactosidase activities. The transferase-catalysed reaction produces exclusively the desired product in beta -1,4-glycosidic linkage whereas the galactosidase-catalysed reactions predominantly form a 1,6-linked disaccharide. With galactosidase, in order to change the regioselectivity, blocking of the 6-position of 3-O-methyl-D-glucose and anomeric modification of the acceptor structure were investigated. Although acetylation of the 6-position of 3-O-methyl glucose catalysed by lipase was successful, the synthesis of the desired disaccharide did not occur. (C) 2001 Elsevier Science B.V. All rights reserved.