Peptidoglycan glycosyltransferases (PGTs) are highly conserved enzymes that catalyze the polymerization of Lipid II to form the glycan strands of bacterial murein. Because they play a key role in bacterial cell wall synthesis, these enzymes are potentially important antibiotic targets; however, their mechanisms are not yet understood. One longstanding question about these enzymes is whether they elongate glycan chains by adding subunits to the anomeric (reducing) end or to the 4-hydroxyl (nonreducing) end. We have developed an approach to test the direction of chain elongation that involves the use of nascent peptidoglycan chains which are blocked at their nonreducing ends. In the presence of the PGT domains of Staphylococcus aureus PBP2, Aquifex aeolicus PBP1A, Escherichia coli PBP1A, or Escherichia coli PBP1B, these blocked substrates react with Lipid II to form longer glycan chains. These results establish that PGTs elongate nascent pepticloglycan chains by the addition of disaccharide subunits to the anomeric (reducing) end of the growing polymer.