ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP-Glc ligand. The role of these amino acids (Glu(194), Ser(212), Tyr(216), Asp(239), Phe(240), Trp(274), and Asp(276)) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoglucose PPase family.