Biodesulfurization is an attractive alternative to hydrodesulfurization for lowering the sulfur content of petroleum products. However, the fuel oils are toxic to microorganisms, which have seriously hindered the application of biodesulfurization. Here, a solvent-tolerant desulfurizing bacterium, Pseudomonas putida DS23, was developed using one of the organic solvent-responsive expression vectors newly constructed for biocatalysis, in which gene expression could be regulated in an organic solvent-dependent fashion. The biodesulfurizing activity of P. putida DS23 could be induced by all the organic solvents used. P. putida DS23 cells induced by n-hexane were able to degrade 56% of 0.5 mM DBT in 12 h in the biphasic reaction containing 33.3% (v/v) n-hexane, while the strain induced by isopropyl beta-D-1-thiogalactopyranoside could only degrade 26% of 0.5 mM DBT. These results suggested that use of the constructed organic solvent-responsive expression vectors can facilitate the biphasic biocatalysis involving organic solvents. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.