Steady-state radiolysis, pulse radiolysis and epr studies, combined with enzyme activity measurements, were carried out on the mechanism by which radical attack, through one-electron oxidation, inactivates xanthine oxidase. Electron transfer to both the N3 and Br2- radical species was used to initiate oxidative damage on the enzyme. Inactivation was found to occur to a greater extent at low than at high pH and is associated with the initial formation of a tryptophanyl radical which converts by a known intramolecular pathway to a tyrosyl radical with a rate constant of 5 x 10(3) S-1. The tyrosyl radical in turn slowly loses around half of its absorbance at an intramolecular rate constant of 350S-1 and is consistent with the establishment of a radical equilibrium with cysteine residue(s). The sequence of reactions could be repeated several times on the same irradiated sample implying that restitution of the implied cysteinyl radical occurs leading to other damage in the protein. N3+Trp/N-->Trp/N-->Tyr/O<-->Cys/S-->?. Epr evidence implies that inactivation of the enzyme from the above sequence of reactions arises in part from alternations to Fe/S center I in the enzyme.