The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.