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Environmental regulations are driving R&D efforts to produce low sulfur fuels, including diesel fuel and gasoline for motor vehicles. Biocatalytic sulfur removal from fuels has potential applicability for producing low sulfur gasoline. Microbial biocatalysts have been identified that can biotransform sulfur compounds found in fuels, including ones that selectively remove sulfur from dibenzothiophene heterocyclic compounds. Most attention is give to the 4S pathway of Rhodococcus, which can remove sulfur from substituted and unsubstituted dibenzothiophenes, including sulfur compounds that hinder chemical catalysis and that resist removal by mild hydrotreatment. Various bioreactor and bioprocess designs are being tested for use with biocatalysts, including recombinant biocatalysts, for use in removing sulfur from fuels and feedstocks within the petroleum refinery stream. With bioprocess improvements that enhance biocatalyst stability, achieve faster kinetics, improve mass transfer limitations, temperature and solvent tolerance, as well as broaden substrate specificity to attack a greater range of heterocyclic compounds, biocatalysis may be a cost-effective approach to achieve the production of low sulfur gasoline. The challenge will be to accomplish these improvements by the time the regulations for low sulfur gasoline and other vehicle fuels go into effect in order to be competitive with emerging nonbiological desulfurization technologies.