We investigated whether apurinic/apyrimidinic (AP/abasic) sites were more frequent in regions of DNA replication in cells and whether their number increased during oxidative stress. DNA fiber spreading and fluorescent immunostaining were used to detect areas of DNA replication and sites of AP lesions in extended DNA fibers. The distribution of AP sites was determined in DNA fibers from vertebrate cells maintained under normal culture conditions or stressed with exogenous H2O2. AP lesions per unit length were enumerated in bulk DNA or at replication sites. The background density of AP sites in DNA fibers was 5.4 AP sites/106 nt, while newly replicated DNA contained 12.9 AP sites/106 nt. In cells exposed to 20 μM H2O2, AP sites in newly replicated DNA increased to 20.8/106 nt. Determinations of AP site density in bulk DNA by fiber analysis or standard slot blot assays agreed to within 10%. Our findings show that the fiber assay not only accurately determines the frequency of AP sites but also shows their distribution. They also reveal that there is increased susceptibility to oxidative damage in DNA regions undergoing replication, which may explain the previously observed clustering of AP sites.—Chastain II, P. D., Nakamura, J., Rao, S., Chu, H., Ibrahim, J. G., Swenberg, J. A., Kaufman, D. G. Abasic sites preferentially form at regions undergoing DNA replication.