Reactive oxygen species (ROS) and the oxidative DNA damage they produce [e.g., 8-oxo-guanine and apurinic/apyrimidinic (AP) sites] have been linked to the pathogenesis of several age-related and chronic diseases. The basal number of AP sites measured in DNA by immuno-slot-blot analysis ranges from 70,000 to 100,000 per genome. We used electron microscopy to determine how AP sites were distributed in isolated DNA fibers from fresh calf thymus and HeLa cell cultures. We observed that AP sites were not equally distributed throughout all the fibers. A small percentage of the analyzed DNA fibers contained a disproportionate amount of the total AP sites in nonrandom groups of 10 to >30 closely spaced in a small region (e.g., 20 AP sites in a 6 kb length of DNA). This finding suggests that genomic sites may differ in their vulnerability to ROS damage, perhaps because of local chromatin structure. Nonrandom AP site formation also suggests that the detrimental effects of ROS in the development of disease may be related not simply to the total number of AP sites present but to how AP sites are distributed along a DNA fiber and, perhaps, to the genomic sites affected.--Chastain, II, P. D., Nakamura, J., Swenberg, J., Kaufman, D. Nonrandom AP site distribution in proliferative cells.