N-acetyl-D-glucosamine (GlcNAc) 2-epimerase catalyzes the interconversion between GlcNAc and N-acetyl-D-mannosamine (ManNAc) that is a precursor of N-acetyl-D-neuraminic acid (NeuAc). Homology search using the sequence of the porcine GlcNAc 2-epimerase as a query revealed that a gene product (Slr1975) of Synechocystis sp. PCC6803 showed significant homology. When the gene of slr1975 was cloned by PCR and expressed in Escherichia coli, the recombinant E. coli showed GlcNAc 2-epimerase activity. This is the first example of the cloning of the gene for GlcNAc 2-epimerase from prokaryotes. GlcNAc 2-epimerase was purified from E. coli overexpressing slr1975, and the enzymatic properties were determined. Molecular weight by SDS-PAGE was 45 kDa, similar to that predicted by the sequence. Km values for GlcNAc and ManNAc were 6.94 mM and 4.76 mM, respectively, and ATP was essential for the activity. Microbial production of NeuAc was carried out using E. coli cells overexpressing GlcNAc 2-epimerase and NeuAc synthetase as enzyme sources. Phosphoenolpyruvate and ATP, required as a substrate or a cofactor of the enzymes, were supplied by the activities of E. coli and Corynebacterium ammoniagenes cells. Starting with 800 mM GlcNAc and 360 mM glucose, NeuAc accumulated at 39.7 mM (12.3 g l(-1)) after 22 h.