In order to understand how beta-(1-->4)-galactosyltransferase recognizes its glycosyl acceptor, substrate specificities were investigated using synthetic 2-acetamido-2-deoxy-D-glucopyranose (N-acetylglucosamine) derivatives in which the 1-, 2-, 3-, 4-, and 6-positions were systematically substituted. The hydroxyl groups at the 3-, 4-, and 6-positions were substituted by fluoride, thiol or hydrogen. For modification of the 2-position, the acetamido group was converted to ethylamino-, N-methylacetamido- and acetyloxy groups. For the anomeric position, several sugar residues were introduced as the aglycon of N-acetylglucosaminide. Galactose transfer assay using synthetic N-acetylglucosamine derivatives indicated that both the acetamido group and the 4-hydroxyl group were essential for binding of N-acetylglucosamine toward the beta-(1-->4)-galactosyltransferase. The assay also showed that the N-acetylglucosamine having a large substitution at the 6-position can be recognized as an acceptor. It is suggested that in this case the bulky substitutent is positioned away from the catalytic site or out of enzyme. Since the 2-acetamido and the 4-hydroxyl group are essential for recognition, the side composed of the 2, 3, and 4-positions may face the acceptor-binding site. (C) 1998 Elsevier Science Ltd. All rights reserved.