A production system of UDP-N-acetylglucosamine (UDP-GlcNAc) was established by using recombinant Escherichia coli and Corynebacterium ammoniagenes in combination. E. coli overexpressed the UDP-GlcNAc biosynthetic genes, glmM, glmU, glk, ppa, ack, and pta, whereas C. ammoniagenes contributed to the formation of UTP from orotic acid. Glucose 1,6-diphosphate (Glc-1,6-P-2), which was required for the activity of phosphoglucosamine mutase involved in UDP-GlcNAc biosynthesis, was supplied by phosphoglucomutase and phosphofructokinase. Starting with orotic acid (65 mM) and glucosamine (400 mM), UDP-GlcNAc accumulated at 11.4 mM (7.4 g l(-1)) after 8 h.