Nicotine and nornicotine are the two main alkaloids that accumulate in Nicotiana tabacum L. (tobacco), and nornicotine is the N-demethylation metabolite of nicotine. Nicotine is synthesized in the root, and probably primarily in the root tip. Both nicotine and nornicotine exist as two isomers that differ from each other by the orientation of H atom at the C-2' position on the pyrrolidine ring. (S)-nicotine is the dominant form in tobacco leaf and the enantiomer fraction of nicotine (EFnic), the fraction of (R)-enantiomer over the total nicotine, is approximately 0.002. Despite considerable efforts to elucidate nicotine and nornicotine related metabolism, a comprehensive understanding of the factors responsible for regulating the variable EF for nornicotine (0.04 to 0.75 ) relative to nicotine has been lacking. The objectives of these investigations were to understand the mechanisms behind the discrepancy. There are three nicotine demethylases reported to be active in tobacco. In vitro recombinant CYP82E4, CYP82E5v2 and CYP82E10 demethylated (R)-nicotine three, ten and ten-fold faster than (S)-nicotine, respectively, and no racemization was observed in either nicotine or nornicotine during demethylation. To confirm these in vitro results, the accumulation and demethylation of nicotine enantiomers throughout the growth cycle and curing process were investigated. Scion stock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf. The results indicate that nicotine consists of 4% of the R enantiomer (0.04 EFnic) when synthesized. However, (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5 and CYP82E10, resulting in an approximate 0.01 EFnic and 0.60 EFnnic in the root. After most of (R)-nicotine is demethylated in root, nicotine and nornicotine are translocated to leaf, where nicotine is further demethylated. Depending on the CYP82E4 activity, an EFnnic of 0.04 to 0.60 is produced and only 0.2% of the remaining nicotine in the leaf is (R)-configuration.