The disaccharide alpha-L-Fuc p-(1-->2)-beta-D-Gal p-O-(CH2)7CH3 (6) is an acceptor for the glycosyltransferases responsible for the biosynthesis of the A and B blood-group antigens. These enzymes respectively transfer GalNAc and Gal in an alpha linkage to OH-3 of the Gal residue in 6. All eight possible O-methyl, epimeric, and amino analogues of 6 having modifications on the target Gal residue were chemically synthesized and kinetically evaluated both as substrates and inhibitors for the A and B glycosyltransferases. The results support earlier findings that both enzymes will tolerate replacement of the hydroxyl groups at the 3 and 6 positions of the Gal residue. Substitution at or replacement of OH-4 of the Gal residue, however abolishes recognition. The 6-O-methyl and 6-amino compounds are substrates for both enzymes while the 3-epimeric (10) and 3-amino (12) compounds are inhibitors. For the B transferase, 10 is a competitive inhibitor with a Ki of 7.8 microM. Attempts to determine a Ki for 12 with the B transferase were unsuccessful because of a complex mode of inhibition. Similarly, both 10 and 12 are potent inhibitors of the A transferase, but the inhibition constants could not be calculated because of a complex mode of inhibition, resembling that for the B transferase. With the A transferase, 12 had an estimated Ki in the 200 nM range.