The stereochemical outcome of oxygen transfer to the sulfur moiety of aryl alkyl sulfides catalyzed by two flavoenzyme monooxygenases has been determined by resolution of sulfoxide product enantionmers on a high-pressure liquid chromatography column [Pirkle, W. H., Finn, J. M. Schreiner, J. L., & Hamper, B. C. (1981) J. Am. Chem. Soc. 103, 3964-3966] containing a 3,5-dinitrobenzoyl-D-phenylglycine chiral stationary phase. With 4-tolyl ethyl sulfide as substrate, cyclohexanone monooxygenase from Acinetobacter produces predominantly the (S)-(-)-sulfoxide (82% S, 18% R), a modest enantioselectivity. In contrast, the flavin adenine dinucleotide (FAD) containing a monooxygenase purified from hog liver microsomes carries out sulfoxidation to yield the (R)-(+)-sulfoxide enantiomer as major product (95% R, 5% S). The presence of the minor sulfoxide enantiomer in each case appears to be due to incomplete chiral processing by each enzyme and not to a competing, achiral, nonenzymic sulfoxidation process. The mammalian FAD-containing monooxygenase also oxygenates the divalent sulfur of the antiarthritic drug sulindac sulfide to yield a single dextrorotatory isomer of the sulfoxide prodrug. Analysis of the chiral outcome of sulfoxidation catalyzed by rat liver microsomes indicated that phenobarbital treatment increases the capacity for S-(-)-oxygenation of 4-tolyl ethyl sulfide, suggesting that the phenobarbital-induced cytochrome P-450 isoenzymes catalyze formation of the (S)-(-)-sulfoxide preferentially, a surmise validated in the following paper [Waxman, D. J., Light, D. R., & Walsh, C. (1982) Biochemistry (following paper in this issue)]. With sulindac sulfide as substrate, though, both control and phenobarbital-induced microsomes catalyze sulfoxidation to yield the same (+)-sulfoxide enantiomer generated by the purified FAD-containing monoxygenase, suggesting a low degree of participation by the cytochrome P-450 isozymes in sulfoxidation of this compound.