Abstract: The relationship between the expression levels and enzymatic activities of 10 CYP2A6 proteins from different source activation systems were investigated in this work. The systems included cells (BEAS-2B cells, HepG2 cells, and macrophages) from human, human liver microsomes from 25 donors of mix gender, human liver microsomes from males, liver microsomes and S9 from animals commonly used in laboratories (male SD rat liver microsomes and male rhesus monkey liver microsomes, male beagle liver microsomes, induced liver S9 of male rats) and homologous recombinase. The expression levels of CYP2A6 protein were determined by western blot assay, and the metabolic kinetic parameters were assessed on the basis of nicotine metabolism by HPLC-MS/MS method. The results showed that:1) CYP2A6 from the 10 activation systems presented clear differences in enzymatic activity. The nicotine concentration significantly reduced when incubating with human liver microsomes from 25 donors of mix gender, homologous CYP2A6 recombinase and liver microsomes of male rhesus monkeys separately, however there was no or weak enzymatic activity in the other 7 activation systems. 2) There existed a certain correlation between the content of CYP2A6 protein and the enzyme activity of CYP2A6. The results of this study offers a useful reference for the selection of metabolic activation system used in the toxicological evaluation of cigarette smoke and aerosols from next generation nicotine products in vitro, and provides in vitro experimental data for understanding the effects of CYP2A6 gene polymorphism on nicotine metabolism.