AIM To identify potential amino acid residues that contribute to different catalytic characteristics of CYP2A6 and CYP2A13 enzymes in nicotine metabolism. METHODSWild type of CYP2A6 and CYP2A13 and their mutants CYP2A6V117A, CYP2A6G164H, CYP2A6I208S, CYP2A6R372H, CYP2A6S465P and CYP2A13A117V, CYP2A13H164G, CYP2A13S208I, CYP2A13H372A and CYP2A13P465S, were subjected kinetic analysis in nicotine 5'-hydroxylation. RESULTS For CYP2A6, substitution of isoleucine 208 to serine caused dramatic kinetic property changes with K_m and V_( max) varied from 62.25 μmol·L~(-1) and 6.53 mol·min~(-1)·moL~(-1) to 345 μmol·L~(-1) and 2.19 mol·min~(-1)·moL~(-1). However, the corresponding serine 208 to isoleucine mutation did not heavily affect the enzyme activity in CYP2A13. The histidine 372 to arginine mutation resulted in a remarkable catalytic efficiency decrease with K_m and V_( max) changes from 26.01 μmol·L~(-1) and 24.51 mol·min~(-1)·moL~(-1) to 148.7 μmol·L~(-1) and 6.11 mol·min~(-1)·moL~(-1) in CYP2A13, but the switching of argenine 372 to histidine did not show expected corresponding crucial influence in CYP2A6 activity. Substitutions on the other positions changed enzyme activities in different rates. CONCLUSION The isoleucine 208 is crucial to human CYP2A6, while the 372 histidine is a key amino acid residue for CYP2A13 in nicotine 5'-hydroxylation.