A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [ nicotinamide adenine dinucleotide ( reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)(-1) h(-1). Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)(-1) h(-1), yielding 87 g l(-1) D-mannitol from 93.7 g l(-1) D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/ NAD(+) ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l(-1) D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)(-1) h(-1) was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells.