A recombinant Escherichia coli strain was developed to produce guanosine 5'-diphosphate (GDP)-L-fucose, donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-D-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25 degrees C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 +/- 0.6 mg l(-1) was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 +/- 0.5 mg l(-1) GDP-L-fucose under the same cultivation condition.