The mechanism of the demethylation of nicotine [3-(1-methyl-2-pyrrolidinyl)pyridine] to nornicotine [3-(2-pyrrolidinyl)pyridine] has been studied in root cultures of Nicotiana alata using nicotine selectively labelled with H-2. Administered [4',4',5',5'-H-2(4)]nicotine was effectively incorporated into [4',4',5',5'-H-2(4)]nornicotine without a significant alteration in the molar fractions of 2H at the four substituted positions, as determined by quantitative H-2 NMR. Incorporation into cotinine-d(2) and myosmine-d(4) was also observed: In contrast; no incorporation into either nornicotine or nicotine was found when [4',4'-H-2(2)]cotinine was administered. Taken together, these findings provide direct evidence that neither of the protons on C-5' of nicotine takes part in the demethylation reaction. (C) 1997 Elsevier Science Ltd. All rights reserved.