Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate. When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity). However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared. A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak. Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate. Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation. Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected. These findings suggest that NeuAc synthase is not present in E. coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.