CMP-Sialic acid synthetase from Neisseria meningitidis 406Y was expressed in Escherichia coli K113 pLysS and produced at 360 U/L. The purified CMP-sialic acid synthetase used both N-acetyl-neuraminic acid (K-m = 0.34 mM) and N-glycolyl-neuraminic acid (K-m = 2.6 mM) as substrates. The recombinant synthetase could be used in a coupled reaction with an alpha-2,3-sialyltransferase to sialylate a lactose derivative in a one-reactor synthesis.