One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h ΠΈ g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/ h ΠΈ g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.