Transient histidine radicals formed in aqueous solutions by oxidation of histidine with a Ti3+/H2O2 Fenton system at pH 2.0 have been studied by EPR using a fast continuous-flow setup and a dielectric ring resonator equipped with a mixing chamber. A histidine peroxy radical with a single EPR line at g = 2.0151 and a histidine cation radical with a complex hyperfine structure and g = 2.0023 have been detected. The hyperfine structure of the latter radical was analyzed by investigating two selectively deuterated histidines and using an EPR simulation and fit program for analysis of the spectra. Isotropic hyperfine coupling constants of two beta-protons, three ring protons, and two nitrogen nuclei have been determined in this way and assigned to a histidine-OH adduct cation radical. Density functional theory (DFT) calculations at the B3LYP and PWP86 levels have been performed on protonated cation radicals of 4-ethylimidazole (as histidine models), yielding isotropic hyperfine coupling constants for three different positions of OH addition. The C5 position for OH addition (a 5-oxohistidine cation radical) is clearly supported by the calculated hyperfine coupling constants. The agreement between DFT and EPR is further improved when hydrogen-bonding interactions to the N1 and C2 protons are introduced in the calculations.