A heterotrimeric flavoprotein-cytochrome c complex fructose dehydrogenase (FDH) of Gluconobacter japonicus NBRC3260 catalyzes the oxidation of d-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The fdhSCL genes encoding the FDH complex of G. japonicus NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for fdhSCL encoding the small, cytochrome c, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of fdhSCL was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of Gluconobacter oxydans and a G. oxydans strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated (TTG)FDH and (ATG)FDH. Membranes of the cells producing recombinant (TTG)FDH and (ATG)FDH showed approximately 20 times and 100 times higher specific activity than those of G. japonicus NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high d-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome c subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.