The enzyme which catalyzes the adenosine triphosphate-dependent addition of L-alanine to uridine diphosphate N-acetylmuramic acid has been purified approximately 100-fold from Staphylococcus aureus. The products of the reaction are uridine diphosphate N-acetylmuramyl-L-alanine, adenosine diphosphate, and inorganic phosphate. In addition to glycine, DL-serine at high concentrations acts as substrate, but D-alanine does not. The enzyme reaction requires NH4+ or K+ besides Mn2+ or Mg2+ and is stimulated markedly by 2-mercaptoethanol.