Phosphoenolpyruvate:uridine-5'-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltranferase catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine with the liberation of inorganic orthophosphate. It was purified to homogeneity from Enterobacter cloacae with the use of UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-Dap, a feedback inihibitor, as a ligand covalenty bound to Sepharose 4B. The evidence suggests that the enzyme is a single polypeptide with a molecular weight of 41,000. The enzyme catalyzes the first committed step in the biosynthesis of bacterial cell wall peptidoglycan. The cytoplasmic end product of this pathway is UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-Dap-D-Ala-D-Ala (see article). UDP-MurNAc-pentapeptide and its precursor, UDP-MurNAc-tripeptide, were found to be effective inhibitiors of the enzyme. The kinetic data suggest a binding site for these inhibitors distinct from the active site. This is consistent with the proposed role for UDP-MurNAc-tripeptide and pentapeptide as negative modulators of the enzyme.