DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3 ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3 blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strandspecific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3 ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2-to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.DNA is intrinsically unstable: it undergoes spontaneous hydrolysis of labile N-glycosyl bonds, as well as oxidation and nonenzymatic methylation at significant rates in vivo (47). These processes are thought to contribute to mutagenesis, carcinogenesis, and aging (2, 3) and are suspected to be amplified by a plethora of environmental pollutants. Reactive oxygen species (ROS) such as superoxide radicals (O 2 Ϫ ) and H 2 O 2 are generated in all aerobic cells (19). Excess production of these ROS by endogenous sources, for example, mitochondria and activated leukocytes, or by exogenous sources such as redox-cycling quinones (75) will exacerbate oxidative damage to cellular DNA (33, 54). The toxicity of these ROS is thought to result at least in part from their conversion to the highly reactive hydroxyl radical by transition metal-catalyzed Fentontype reactions (4,25). This radical is also formed by the interaction of ionizing radiation with water, e.g., within cells (78), and is known to induce a broad spectrum of DNA lesions, including base and sugar modifications, base loss, and DNA strand breaks (8, 9). The most frequent lesions detected in cells exposed to ionizing radiation are oxidized apurinic/apyrimidinic (AP) (abasic) sites (27) and single-strand breaks, most of which are te...