Binding of various C6 galactose derivatives to Ricinus communis agglutinin (RCA I) was studied in order to better understand the structural elements involved in lectin-sugar interactions. The inability of D-galacturonic acid to inhibit the hemagglutination of red blood cells by RCA I, at low concentrations, is shown to be due to intramolecular hydrogen bonding between the hydrogen atom of the C4 hydroxyl group and one of the oxygen atoms of the carboxylate anion of D-galacturonic acid and not to the negative charge and/or the steric size of the C6 carboxylate as previously suggested. Thus it is unequivocally shown that during the binding of D-galactose to RCA I, the C4 hydroxyl group of D-galactose serves as the hydrogen bond donor. It is also shown that a negatively charged substituent at the C6 atom of D-galactose, as in D-galactose-6-sulfate, does not inhibit the binding of the modified D-galactose molecule to RCA I, whereas a positively charged group, as in protonated 6-amino-6-deoxy-D-galactose, dramatically inhibits the binding.