Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS-PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The K(m) values of Neu5Ac-9-phosphate synthase were 35 microM for N-acetylmannosamine-6-phosphate and 100 microM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn(2+), Fe(2+), and Mg(2+) being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.