This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species. U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups. In an incubation for 22 h, 1.2 mM peroxyl radicals was generated and each cell acquired 1.5 x 10 8 -OOH groups. These groups were located on the cell proteins; no lipid peroxidation was detected. The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals. There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins. The half-life of protein hydroperoxides in cell suspensions was approx. 4 h at 37 °C. Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species.