Reactive oxygen species (ROS) induce 8–hydroxy–2'-deoxyguanosine (8-OHdG) formation, which has been proposed as a key biomarker relevant to carcinogenesis. 8-OHdG has been induced in a number of different ways, most often without knowledge of the specific type and amount of ROS generated. We have measured 8-OHdG formation in calf thymus DNA exposed to ionizing radiation under conditions generating either hydroxyl radicals (OH·), superoxide anions (O2–) or both. Additionally, we investigated the relationship between the scavenger effect of the drug 5-aminosalicylic acid (5-ASA) and increasing OH· exposure toward 8-OHdG formation. The effect of this drug was compared to those of the physiological scavengers ascorbate and reduced glutathione (GSH). We found that OH· generated 8-OHdG in a dose-dependent manner, whereas O2– did not cause 8-OHdG formation. 5-ASA, ascorbate and GSH all acted as hydroxyl radical scavengers, although with different concentration-effect curves, emphasizing the importance of using relevant pharmaco-/physiological concentrations in studies focusing on therapeutic applications of scavengers. The scavenger effect of 5-ASA at concentrations ≥ 0.1 mM was similar at 30 and 100 Gy radiation, i.e. within a wide range of OH- exposure, which is useful information considering clinical applications where the exact amount of ROS formed is unknown. Both 5-ASA and ascorbate at low concentrations (≤ 0.1 mM) were less efficient in preventing 8-OHdG formation from X-ray generated OH· than was shown in a previous comparable study using light as the source of ROS. This differentiation probably reflects variations in both number and type of ROS formed in the two systems.