An alternative approach to water-based biodesulfurization systems was examined employing organic solvent-based reaction media. A gram-position microorganism, FE-9, was developed to function biocatalytically against organic sulfur-containing heterocycles in 100% dimethylformamide. Strain FE-9 converted dibenzothiophene to biphenyl and hydrogen sulfide under a hydrogen atmosphere, and to biphenyl, hydroxybiphenyl and sulfate under an air atmosphere. Thianthrene was converted to benzene and hydrogen sulfide under a hydrogen atmosphere, and to benzene and sulfate under an air atmosphere. The bioconversion of sulfur-containing heterocycles to aromatic hydrocarbons and hydrogen sulfide, and the oxidation of sulfide to sulfate under air, appears to represent two different biochemical events occurring in dimethylformamide. These studies indicate a strong potential for the development of biocatalysts that selectively abstract organosulfur from high-sulfur containing fossil fuels in an organic solvent-based reaction medium.