The acetylation mechanisms of several selected typical substrates from experiments, including arylamines and arylhydrazines, are investigated with the density functional theory in this paper. The results indicate that all the transition states are characterized by a four-membered ring structure, and hydralazine (HDZ) is the most potent substrate. The bioactivity for all the compounds is increased in a sequence of PABA ≈ 4-AS < 4-MA < 5-AS ≈ INH < HDZ. The conjunction effect and the delocalization of the lone pairs of N atom play a key role in the reaction. All the results are consistent with the experimental data.