Nicotine and nornicotine are two of the dominant alkaloids found in tobacco (N. tabacum L.), and known precursors to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN).  In tobacco nicotine is N-demethylated to nornicotine by nicotine demethylase enzymes, which belong to a family of cytochrome P450 (CYP) proteins.  CYP82E4 (E4) and CYP82E5 (E5) are two nicotine demethylases which have been reported to be active in tobacco.  Both nicotine and nornicotine have two enantiomers which differ from each other at the 2' position on the pyrrolidine ring.  Naturally occurring nicotine is almost exclusively (-)-2'-S-nicotine, and (+)-2'-R-nicotine accounts for less than 1% of total nicotine, while after demethylation 4 to 75% of nornicotine is (+)-2'-R-nornicotine and the percentage varies among lines and tissues.  The reason for this high and variable percentage of (+)-2'-R-nornicotine resulting from low and constant (+)-2'-R-nicotine substrate is unknown.  One hypothesis is that (+)-2'-R-nicotine is preferentially demethylated to (+)-2'-R-nornicotine.  The objective of this study was to investigate the enantioselectivity of the E4 demethylase, the enzyme reported to be responsible for the majority of nicotine demethylation occurring in the plant.  Using microsomes prepared from yeast over-expressing the E4 gene as a source of demethylase enzyme, the kinetics of enzyme enantioselectivity were determined by measuring demethylase activity at several concentrations of 2'-14C- R, S or racemic nicotine.  These enzyme assays demonstrated that the E4 enzyme demethylated (+)-2'-R-nicotine 3-fold faster than (-)-2'-S-nicotine, and exhibited a 10-fold lower Km (Michaelis constant) for (+)-2'-R-nicotine than (-)-2'-S-nicotine, which means E4 has greater affinity for (+)-2'-R-nicotine.  Our results clearly demonstrate the enantioselective preference of the E4 demethylase for (+)-2'-R-nicotine, which may partially explain the high percentage of (+)-2'-R-nornicotine in the plant produced from low (+)-2'-R-nicotine substrate.