Two different fucosyltransferases (Fuc-Ts) have been isolated from human milk, an α1-3 Fuc-T and an α1-3/4 Fuc-T, for mapping of their acceptor binding sites. Kinetic studies employing a series of monodeoxygenated and modified Galβ1→4Glc-NAcβOR and Galβ1 → 3GlcNAcβOR acceptor substrates showed that modifications are tolerated at every hydroxyl group in these substrates except for 6-OH of galactose and 3- or 4-OH of N-acetylglucosamine. Deoxygenation at these positions rendered these compounds inactive as both substrates and inhibitors. These essential hydroxyl groups, which are required for recognition of the substrates, are identical to the key polar groups that have previously been reported for cloned FucTs III, IV and V.