The gene encoding glucose-fructose oxidoreductase (gfo) from Zymomonas mobilis was cloned in Escherichia coli and sequenced. An open reading frame of 439 amino acids encoded a protein of 49 kDa. A leader sequence of 52 amino acids preceded the N-terminal sequence of the enzyme, indicating cleavage of the precursor protein at an Ala-Ala site to give rise to an active form of the enzyme of 43 kDa. Processing of the glucose-fructose oxidoreductase leader sequence, although not complete, was demonstrated in an in vitro translation system. The two Z. mobilis promoters of the gfo gene show considerable homology to other highly expressed Z. mobilis genes (pdc, adhB, gap, and pgk) as well as to the E. coli consensus sequence. Although translation of the gfo gene was demonstrated in vitro in an E. coli S30 coupled transcription-translation system, a functional stable protein was not produced in the E. coli clone. However, the gfo gene cloned into a shuttle vector was shown to overexpress glucose-fructose oxidoreductase to levels of up to 6% of the soluble protein in Z. mobilis.