Heavy-atom isotope effects for the N-demethylation of nicotine have been determined in vivo in static-phase biosynthetically incompetent plant cell cultures of Nicotiana species. A 2 H kinetic isotope effect of 0.587 and a 15 N kinetic isotope effect of 1.0028 were obtained. An identical 15 N kinetic isotope effect of 1.0032 was obtained for the nicotine analogue, N-methyl-2-phenylpyrrolidine. The magnitude of the 15 N heavy-atom isotope effect indicates that the fission of the CN bond is not rate limiting for demethylation. The theoretical calculation of heavy-atom isotope effects for a model of the reaction pathway based on cytochrome P450 best fits the measured kinetic isotope effect to the addition of hydroxyl ion to iminium to form N-hydroxymethyl, for which the computed 2 H- and 15 N kinetic isotope effects are 0.689 and 1.0081, respectively. This large inverse 2 H kinetic isotope effect is not compatible with the initial abstraction of the H from the methyl group playing a significant kinetic role in the overall kinetic limitation of the reaction pathway, since computed values for this step (4.54 and 0.9995, respectively) are inconsistent with the experimental data.