A homologous series of alkyl-substituted p-tolyl sulfides have been synthesized and evaluated as in vitro, isozyme-selective substrate probes for the microsomal flavin containing monooxygenases. Straight-chain and branched-chain alkyl homologs were metabolized to the corresponding (R)- and (S)-sulfoxides which were analyzed by chiral phase high-performance liquid chromatography. Initial studies demonstrated that the stereochemical composition of alkyl p-tolyl sulfoxides generated by FMO2, purified from rabbit lung, was a function of the degree of steric crowding about the prochiral center. In contrast, purified rabbit liver FMO1 formed the (R)-sulfoxide from the n-alkyl series of substrates in a highly stereoselective manner (> 90%). Similar results were obtained with these two rabbit cDNAs expressed in E. coli. In contrast to rabbit FMO1 and FMO2, a characteristic feature of catalysis by cDNA-expressed rabbit FMO3 was the lack of stereoselectivity observed for formation of methyl p-tolyl sulfoxide. Collectively, these data demonstrate that the stereochemical composition of sulfoxides generated from the n-alkyl series of sulfides is isozyme-dependent. Metabolism of methyl p-tolyl sulfide by detergent-solubilized hepatic microsomes from a wide variety of experimental animals yielded predominantly (R)- methyl p-tolyl sulfoxide, which, at least in rabbit liver, is indicative of catalysis dominated by FMO1. However, solubilized human and macaque liver preparations catalyzed this reaction in a relatively non-stereoselective manner. Macaque liver FMO was purified and the metabolite profile generated from the n-alkyl p-tolyl sulfides was found to be most similar to rabbit FMO3. Moreover, antibodies directed against macaque liver FMO selectively reacted with rabbit FMO3 and a microsomal protein expressed in adult human, but not fetal human liver, adult human kidney or adult human lung. Therefore, an FMO isoform expressed selectively in adult primate liver has catalytic and immunochemical properties consistent with its classification in the FMO3 family.