The main focus in this task is the application of desulfurization enzymes to remove sulfur from crude oil, diesel fuel, or resid precursors for needle cokes. The most important question to be answered is how to utilize the Rhodococcus desulfurization complex in a nonaqueous solvent or medium, such as the oil itself. Successful application of nonaqueous enzymology to this problem will involve finding ways to stabilize the active conformations of the enzymes and provide for easy recovery of the enzymes, perhaps in an immobilized-enzyme packed-bed reactor. Understanding the desulfurization activity will require that they determine the regulatory and mechanistic properties of the enzymes. In the previous period, methods were developed for recovering larger amounts of the metabolic intermediates that would at the same time preserve all the labeling from oxygen isotopes by preventing exchange and avoiding displacement during derivatization for the gas chromatography/mass spectroscopy analysis. In this period, methods were investigated for conversion of the sulfonate and the sulfite trapped on the resin to alkyl derivatives that could by analyzed by GC/MS and GC/Fourier transform infrared to determine the oxygen isotopic labeling. This information is needed to complete the understanding of the last steps occurring in the complex desulfurization system in the bacterium. The methods employed included treatment with diazomethane and trimethyloxonium tetrafluoroborate.