Generation of peroxide groups in proteins exposed to a wide variety of reactive oxygen species requires an initial formation of protein carbon-centred or peroxyl free radicals, which can be reduced to hydroperoxides. Both protein radicals and protein hydroperoxides are capable of oxidizing important biomolecules and thus initiate biological damage. In this study, we investigated the inhibition of protein hydroperoxide formation by ascorbate and GSH in irradiated HL-60 cells. Incubation of HL-60 cells with Asc in the presence of ascorbate oxidase resulted in the accumulation of intracellular Asc. The intracellular Asc levels were lowered by irradiation. Exposure of HL-60 cells to increasing irradiation doses resulted in increasing accumulation of protein peroxides in the cells. A significant decrease in intracellular protein hydroperoxides was noted when the cells were treated with Asc before irradiation. A dose-dependent protective effect of Asc was observed. Asc also provided a strong protection from radiation-generated protein hydroperoxides. Protein peroxidation was inhibited in cells with enhanced levels of GSH and increased when the intracellular GSH concentration was reduced.