Nornicotine is the demethylation product of nicotine and the precursor of tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) in tobacco (Nicotiana tabacum L.). There is an inconsistent enantiomer fraction (EF) of nornicotine reported in the literature. The objective of this study was to explore possible mechanisms to account for the variable EF(nnic) in tobacco. A survey of tobacco with different demethylating capabilities confirmed that there was variable EF(nnic). Experiments of induction and inhibition of the major nicotine demethylase CYP82E4 activity in tobacco demonstrated that CYP82E4 selectively demethylated (S)-nicotine and resulted in different EF(nnic) in tobacco leaves. Results from plants with silenced demethylases by RNAi suggested that other demethylases selectively used (R)-nicotine and resulted in high EF(nnic). In summary, enantioselective demethylation likely plays an important role in contributing to a large and variable EF(nnic) observed in tobacco.