Ricin B-chain (RTB) is a galactose-specific lectin that folds into two globular domains, each of which binds a single galactoside. The two binding sites are structurally similar and both contain a conserved tripeptide kink and an aromatic residue that comprises a sugar-binding platform. Whereas the critical RTB residues implicated in lectin activity are conserved in domain 1 of Ricinus communis agglutinin (RCA) B-chain, the sugar platform aromatic residue Tyr-248 present in domain 2 of RTB is replaced by His in RCA B-chain. In this study, key residues in the vicinity of the binding sites of the Ricinus lectin B-chains were altered by site-directed mutagenesis. The recombinant B-chains were produced in Xenopus oocytes in soluble, stable, and core-glycosylated forms. Both sites of RCA B-chain must be simultaneously modified in order to abolish lectin activity, indicating the presence of two independent, functional binding sites/molecule. Activity associated with the domain 2 site of RCA B-chain is abrogated by the conversion of Trp-258 to Ser. Moreover, the domain 2 site appears responsible for a weak binding interaction recombinant RCA B-chain with GalNAc, not observed with native tetrameric RCA. Finally, the introduction of His at position 248 of RTB severely disrupts but does not abolish GalNAc binding.