A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures, and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common polymorphisms may affect the structure of this protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the phosphate-binding loop was found using our models and will be discussed. Quantum mechanical calculations on the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP show that the reaction coordinates differ for these two compounds, but the activation barrier separating the reactant from the product are both low. The results of this study suggest that common polymorphisms in human NAT2 are distant from the active site and are more likely to destabilize the enzyme than affect catalysis. Additionally, the quantum mechanical calculations show that the observed differences in mutagenic activity between N-OH MeIQx and N-OH PhIP are not related to their acetylation reaction with NAT.