The oxidation of (2'S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5'-carbon position (A) is favored over oxidation at the methyl carbon (CMe) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5' oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5'. Neither protonation of the pyrrolidine nitrogen (N1') nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a (2)H in either the C5' or the CMepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2-2.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5' or the CMe positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for (2)H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5' or the CMe positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction.