Neoglycoproteins containing various monosaccharides and disaccharides were prepared by modifying bovine serum albumin (BSA) with thioglycosides using amidination or reductive alkylation [Lee, Y. C., Stowell, C. P., & Krantz, M. J. (1976) Biochemistry 15, 3956-3963; Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. The binding specificity of the rabbit liver galactose/N-acetylgalactosamine specific lectin was studied with these neoglycoproteins by assaying their ability to inhibit 125I-labeled asialoorosomucoid binding to the isolated lectin or to the crude plasma membrane. The following observations were made: (1) All the galactose-containing neoglycoproteins (except two) had a similar inhibitory effect. (2) BSA derivatives containing glucose, with the sole exception of [Glc--S--CH2C(= NH)--NH]n-BSA, were poorer inhibitors than the galactose-containing BSA's, and the axial 4-OH of galactose appears to participate in the binding. (3) The binding site of the lectin for the C-6 region of galactose is apparently spacious, since a galactose substituted at the 6-OH with another monosaccharide can still be bound. (4) A negatively charged group at C-6 of galactose interfered with the binding of the lectin. (5) A bulky aglycon in an alpha-D-galactopyranoside considerably decreased the inhibitory power. For explanation of these results, it is proposed that the lectin possesses at least one negatively charged group at the binding site and the presence of this group is directly responsible for inability of the sialylated (negatively charged), native glycoproteins to bind to the mammalian hepatic lectin.