The reaction mechanism of the DNA (cytosine-5)-methyltransferase-catalyzed cytosine methylation was investigated using ab initio quantum mechanical (at the MP2/6-31+G*//HF/6-31+G* and MP2/6-31+G*//HF/3-21+G* levels) and density functional theory calculations (Becke3LYP/6-31+G*) in the gas phase and in solution. The effects of aqueous solvation on the reaction energies were included by using an isodensity surface-polarized continuum model. The quantum mechanical model consisted of 1-methylcytosine (the model of the target cytosine), methylthiolate (the model of the side chain of the catalytic cysteine), and trimethylsulfonium (the model of the methyl-donating AdoMet). In addition, an approach is presented to estimate the pK(a) of the cytosine N3 in the reaction intermediates and transition states and on the calculated reaction profiles. The approach involves calculation of the gas-phase proton affinities and solvation energies of the neutral and protonated forms of the molecules using ab initio quantum mechanical and continuum solvation methods. The calculated aqueous-phase proton affinities were calibrated using a set of 13 nitrogen acids with pK(a) values from 0.5 to 17.5. The correlation coefficient (r(2)) between the calculated aqueous proton affinities and the experimental pK(a)＇s was 0.988. During the attack of methylthiolate on C6 of the cytosine, the pK(a) of the N3 atom of the cytosine was calculated to increase from 5 to 17. In the subsequent reaction step, where C5 of the cytosine is methylated, the pK(a) of N3 drops from 17 to 5. The protonation and deprotonation of the N3 atom was calculated to catalyze the two reaction steps. It seems likely that in the DNA (cytosine-5)-methyltransferase-catalyzed cytosine methylation proton transfers take place between Glu119 of the active site and N3 of the cytosine. The implications of the model calculations for the DNA (cytosine-5)-methyltransferase-catalyzed reactions are discussed.