The enzymatic transglucosylation of three kinds of α-glucosidase (from rat intestine, rice seed and Aspergillus) with lactose as an acceptor was studied. We found out that the three a-glucosidases were capable of synthesizing trisaccharide, which corresponds to glucosyl lactoside (O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranosyl α-D-glucopyranoside) on gas-liquid chroma-tographic analysis, the same as the case of cyclomaltodextrin glucanotransferase (CGTase) reported. The maximum yield of the trisaccharide (3.0%) was obtained with rat intestinal a-glucosidase, and the other two a-glucosidases gave same yield (2.3%). β-Galactosidase converted the trisaccharide into the disaccharide which corresponds to neotrehalose on GLC analysis, and galactose, α-Glucosidase from rice seed also hydrolyzed the trisaccharide into lactose and glucose. These results suggest that the trisaccharide is glucosyl lactoside, and it is the common transglucosylation product of all α-glucosidases used in this study. Neotrehalose was also observed in all the reaction mixture. The α-glucosidases seem to recognize the anomeric hydroxyl group of lactose as a transglucosylation site as well as CGTase.