3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the condensation reaction between phosphoenolpyruvate and D-arabinose 5-phosphate (D-A5P) in a key step in lipopolysaccharide biosynthesis in Gram-negative bacteria. The KDO8P synthase from Neisseria meningitidis was cloned into Escherichia coli, overexpressed and purified. A variety of D-A5P stereoisomers were tested as substrates, of these only D-A5P and l-X5P were substrates. The Asn59Ala mutant of N. meningitidis KDO8P synthase was constructed and this mutant retained less than 1% of the wild-type activity. These results are consistent with a catalytic mechanism for this enzyme in which the C2 and C3 hydroxyl groups of D-A5P and Asn59 are critical.