The waaJ, waaT, and waaR genes encode alpha-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS alpha-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS alpha-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in k(cat) and corresponding increases in K(m)(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His(6)-WaaT, His(6)-WaaJ showed a significantly decreased k(cat) and increased K(m)(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications.