In conjunction with asymmetric chemical syntheses and spectral, chiroptical, chromatographic and stereochemical correlation methods, we have developed procedures for the quantification of sulfoxide enantiomers and tertiary amine N-oxide diastereomer metabolites arising from the action of the adult human liver and other flavin-containing monooxygenases (FMOs). The parallel nature of the metabolic in vitro-in vivo studies and the use of chemical model oxidation systems allowed us to identify the FMO isoform involved. We investigated the enantioselective S-monooxygenation of cimetidine and the diastereoselective tertiary amine N-1'-oxygenation of (S)-nicotine as stereoselective functional probes of adult human liver FMO action. In both cases, the majority of evidence points to adult human liver FMO3 as the principal enzyme responsible for cimetidine S-oxygenation and (S)-nicotine N-1'-oxygenation in vitro and in vivo. The excellent agreement between the absolute configuration of the major cimetidine S-oxide and (S)-nicotine N-1'-oxide metabolites isolated from human urine and the major metabolite formed in the presence of adult human liver microsomes suggests that in vitro hepatic preparations may serve as a useful model for the in vivo condition. Further, that adult human liver cDNA-expressed FMO3 in Escherichia coli also gave the same absolute stereoselectivity (i.e. for (S)-nicotine N-1'-oxygenation) confirms the identity of the monoxygenase in vivo. Although we cannot rule out the involvement of minor contributions of cytochrome P-450 monooxygenases in cimetidine and (S)-nicotine oxidation, the majority of the data support the fact that cimetidine S-oxygenation and (S)-nicotine N-1'-oxygenation are stereoselective functional probes of adult human liver FMO3 activity. Finally, because the stereochemistry of the principal metabolite of cimetidine and (S)-nicotine in small experimental animals is distinct from that observed in humans, it is likely that species variation in predominant FMO isoforms exist and this may have important consequences for the choice of experimental animals in human preclinical drug design and development programs.