The influence of N7 protonation on the mechanism of the N-glycosidic bond hydrolysis in 2'-deoxyguanosine has been studied using density functional theory (DFT) methods. For the neutral system, two different pathways (with retention and inversion of configuration at the C1' anomeric carbon) have been found, both of them consisting of two steps and involving the formation of a dihydrofurane-like intermediate. The Gibbs free energy barrier for the first step is very high in both cases (53 and 46 kcal/mol for the process with inversion and with retention, respectively). However, the N7-protonated system shows a very different mechanism which consists of two steps. The first one leads to the formation of an oxacarbenium ion intermediate, with a Gibbs free energy barrier of 27 kcal/mol, and the second one corresponds to the nucleophilic attack of the water molecule to the oxacarbenium ion and takes place with a barrier of 1.3 kcal/mol. Thus, these results agree with a stepwise SN1 mechanism (DN*AN), with a discrete intermediate formed between the leaving group and the nucleophile approach, and show that N7 protonation strongly catalyzes the hydrolysis of the N-glycosidic bond, making the guanine a better leaving group. Finally, kinetic isotope effects have been calculated for the protonated system, and the results obtained are in very good agreement with experimental data for analogous systems. © 2007 American Chemical Society.