Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower kcat values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased Km values and diminished kcat values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.