The reaction of hydroxyl radical (^•OH) with the nucleic acid base analogue 2-thiouracil (1) has been studied by pulse radiolysis experiments and DFT. The generic intermediate radicals feasible for the ^•OH reactions with 1, namely, one electron oxidation product (1^•+), ^•OH-adducts (3^•, 4^•, and 5^•), and H-abstracted radicals (6^• and 7^•), were characterized by interpreting their electronic and structural properties along with calculated energetics and UV–vis spectra. Pulse radiolysis experiments showed that the transient formed in the reaction of ^•OH with 1 in water at pH 6.5 has λ_max at 430 nm. A bimolecular rate constant, k₂ of 9.6 × 10⁹ M^–1 s^–1, is determined for this reaction via competition kinetics with 2-propanol. The experiments suggested that the transient species could be a dimer radical cation 2^•+, formed by the reaction of 1 with the radical cation 1^•+. For this reaction, an equilibrium constant of 4.7 × 10³ M^–1 was determined. The transient formed in the reaction of 1 with pulse radiolytically produced Br₂^•– at pH 6.5 as well as Cl₂^•– at pH 1 has also produced λ_max at 430 nm and suggested the formation of 2^•+. The calculated UV–vis spectra of the transient species (1^•+, 3^•, 4^•, 5^•, 6^•, and 7^•) showed no resemblance to the experimental spectra, while that of 2^•+ (λ_max = 420 nm) agreed well with the experimental value and thus confirmed the formation of 2^•+. The 420 nm peak was due to σ → σ* electronic excitation centered on a 2-center–3-electron (2c–3e) sulfur–sulfur bond [−S∴S−]. 2^•+ is the first reported example of a dimer radical cation in a pyrimidine heterocyclic system. Further, 5-C and 6-C substituted (substituents are −F, −Cl, −NH₂, −N(CH₃)₂, −OCH₃, −CF₃, −CH₃, −CH₂CH₃, n-propyl, phenyl, and benzyl) and 5,6-disubstituted 2-thiouracil systems have been characterized by DFT and found that the reaction (1 + 1^•+ → 2^•+) is exergonic (1.12–13.63 kcal/mol) for many of them.