Mannitol dehydrogenase (MDH) from Rhodobacter sphaeroides Si4 was overproduced by constructing a strain that overexpresses the MDH gene and by producing high cell concentrations via fed-batch cultivation in a bioreactor. With the gene of mannitol dehydrogenase (mtlK) cloned into the expression vector pKK223-3 expression of MDH in Escherichia coli was obtained, but the specific enzyme activity was lower than in R. sphaeroides Si4. In order to overexpress mtlK in R. sphaeroides, plasmid pAK82 was constructed by cloning a DNA fragment carrying mtlK into the broad-host-range expression vector pRK415. When pAK82 was introduced into R. sphaeroides Si4 the specific mannitol dehydrogenase activity in the strain obtained was 0.48 unit (U)mg-1,3.4-fold higher than in the wild type. In this way the enzyme yield from cultivation in a bioreactor could be improved from 110 Ul-1 to 350 Ul-1. A further increase in productivity was obtained by fed-batch cultivation of R. sphaeroides Si4 [pAK82]. Using this cultivation method an optical density of 27.6 was reached in the bioreactor, corresponding to a dry mass of 16.6 g l-1. Since MDH formation correlated with biomass production, the MDH yield could be raised to 918 Ul-1, an 8.3-fold increase in comparison to batch cultivation of the wild-type strain.