N'-nitrosonornicotine (NNN) is a potent and organ-specific carcinogen found in tobacco and tobacco smoke in substantial amounts. The aim of this study was to investigate the variations in NNN enantiomeric composition in nicotine demethylase mutants. A gas chromatography/thermal energy analyzer method using two columns in series was developed to separate the enantiomers of NNN, N′-nitrosoanabasine, and N′-nitrosoanatabine. Tobacco lines with different combinations of three nicotine demethylases inhibited were grown in the field. Air-cured leaves were analyzed for the concentration and enantiomeric composition of nicotine, nornicotine and NNN. In mutant lines, the concentration of NNN ranged from 0.44 μg g-1 to 13.63 μg g-1 and the percentage of (R)-NNN of total NNN varied from 7% to 69%. The measured NNN had the same enantiomeric composition as nornicotine, rather than nicotine, even when the level of nornicotine was reduced to 0.6% of nicotine in a triple mutant line. The pattern of the enantiomeric composition of nicotine, nornicotine, and NNN demonstrated that the direct formation of NNN from nicotine, if it occurs, is negligible in air-cured tobacco. Since (S)-NNN is dominate form in smokeless tobacco and more carcinogenic than its R form, the reduction of (S)-nornicotine should be a priority for the reduction of NNN.