Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolism enzymes that catalyze the detoxification of arylamines by N-acetylation and the bioactivation of N-arylhydroxylamines by O-acetylation. Endogenous and recombinant mammalian NATs with high specific activities are difficult to obtain in substantial quantities and in a state of homogeneity. This paper describes the overexpression of human wild-type NAT2 as a dihydrofolate reductase fusion protein containing a TEV protease-sensitive linker. Treatment of the partially purified fusion protein with TEV protease, followed by chromatographic purification, afforded 2.8 mg of homogeneous NAT2 from 2 L of cell culture. The kinetic specificity constants (kcat/Km) for N-acetylation of arylamine environmental contaminants, some of which are associated with bladder cancer risk, were determined with NAT2 and NAT1. The NAT1/NAT2 ratio of the specificity constants varied almost 1000-fold for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents. 2-Alkyl substituents depressed N-acetylation rates but were more detrimental to catalysis by NAT1 than by NAT2. 3-Alkyl groups caused substrates to be preferentially N-acetylated by NAT2, and both 4-methyl- and 4-ethylaniline were better substrates for NAT1 than NAT2. NMR-based models were used to analyze the NAT binding site interactions of the alkylanilines. The selectivity of NAT1 for acetylation of 4-alkylanilines appears to be due to binding of the substituents to V216, which is replaced by S216 in NAT2. The contribution of 3-alkyl substituents to NAT2 substrate selectivity is attributed to multiple bonding interactions with F93, whereas a single bonding interaction occurs with V93 in NAT1. Unfavorable steric clashes between 2-methyl substituents and F125 of NAT1 may account for the selective NAT2-mediated N-acetylation of 2-alkylanilines; F125 is replaced by S125 in NAT2. These results provide insight into the structural basis for the substrate specificity of two NATs that play major roles in the biotransformation of genotoxic environmental arylamines.