Guanine (Gua), among purines, is a preferred oxidation/nitration target because of its low one-electron redox potential. The reactive oxygen/nitrogen species peroxynitrite (ONOO⁻), produced in vivo by the reaction between nitric oxide (^•NO) and superoxide radical (O₂^•‒), is responsible for several oxidative modifications in biomolecules, including nitration, nitrosation, oxidation, and peroxidation. In particular, the nitration of Gua, although detected, as well as its reaction kinetics have been seldom investigated. Thus, we studied the concentration- and temperature-dependent formation of 8-nitroguanine (8-NitroGua) in phosphate buffer (pH 7.40) using stopped-flow spectrophotometry. Traces showed a biexponential behavior, with best-fit rate constants: k_fast = 4.4 s⁻¹ and k_slow = 0.41 s⁻¹ (30 °C, 400 μM both Gua and ONOO⁻). k_fast increased linearly with the concentration of both reactants whereas k_slow was concentration-independent. Linear regression analysis of k_fast as a function of Gua and ONOO⁻ concentration yielded values of 2.5–6.3 × 10³ M⁻¹s⁻¹ and 1.5–3.5 s⁻¹ for the second-order (slope) and first-order (ordinate) rate constants, respectively (30 °C). Since ONOO⁻ is a short-lived species, its decay kinetics was also taken into account for this analysis. The 8-NitroGua product was stable for at least 4 h, so no spontaneous denitration was observed. Stopped-flow assays using antioxidants and free-radical scavengers suggested a mixed direct/indirect reaction mechanism for 8-NitroGua formation. Gua nitration by ONOO⁻ was also observed in the presence of physiologically relevant CO₂ concentrations. The reaction product identity, its yield (∼4.2%, with 400 μM ONOO⁻ and 200 μM Gua), and the reaction mechanism were unequivocally determined by HPLC-MS/MS experiments. In conclusion, 8-NitroGua production at physiologic pH reached significant levels in a few hundred milliseconds, suggesting that the process might be kinetically relevant in vivo and can likely cause permanent nitrative damage to DNA bases.