N-Acetylneuraminate (NeuAc) synthase, which catalyzes NeuAc synthesis by condensation of N-acetyl-D-mannosamine (ManNAc) and phosphoenolpyruvate (PEP), was purified from a cell extract of Escherichia coli K1-M12 to electrophoretically homogeneity by serial column chromatographies. The molecular weight of native enzyme was estimated to be 106,000 by gel filtration. After denaturation in sodium dodecyl sulfate, the molecular weight was reduced to 52,000, indicating the existence of 2 identical subunits. The optimum pH was 7.5 and the stable pH range was 7.0 to 10.0. The enzyme was thermostable up to 30 degrees C. No metal ion was required for the enzyme activity. SH-inhibitors such as p-chloromercuribenzoic acid and mercury chloride were potent inhibitors. The K(m) for ManNAc and PEP were 5.6 mM and 0.04 mM, respectively.