The chemical synthesis and chromatographic separation of cis- and trans-(S)-nicotine N-1'-oxide diastereomers have allowed the development of methods for the quantification of (S)-nicotine N-1'-oxides during in vitro and in vivo metabolic studies. The metabolism of (S)-nicotine was investigated in the presence of microsomes, cDNA-expressed and highly purified flavin-containing monooxygenase (FMO) from pig liver, human liver, and rabbit lung. For comparison, the N-1'-oxidation of (S)-nicotine in the presence of the cytochrome P450 2B1 from rat liver, cytochrome P450 2B10 from mouse liver, and cytochrome P450 4A2 from rabbit lung was examined. The ratio of trans:cis (S)-nicotine N-1'-oxide formation for pig liver FMO1 (form 1) was 57:43. In contrast, cDNA-expressed adult human liver FMO3 (form 3) and rabbit lung FMO2 formed solely trans-(S)-nicotine N-1'-oxide. Of the cytochrome P450 enzymes examined, formation of (S)-nicotine N-1'-oxide occurred with a mean trans:cis ratio of 82:18. The stereoselectivity of (S)-nicotine N-1'-oxide formation was investigated by examining the urine of 13 healthy male smokers studied on a protocol which included free-smoking, intravenous infusion of (S)-nicotine-d2 and dermal patch administration of (S)-nicotine-d0. During cigarette smoking or administration of intravenous or transdermal (S)-nicotine, only the trans diastereomer of (S)-nicotine N-1'-oxide was observed in the urine. That the trans-(S)-nicotine N-1'-oxide metabolite was not appreciably reduced or oxidized further was investigated with infusion studies of (S)-nicotine-d2N-1'-oxide.(ABSTRACT TRUNCATED AT 250 WORDS)