When liver, kidney, lung and heart DNA preparations of untreated Sprague-Dawley rats of different ages (3 days-10 months) were analyzed for the possible presence of covalent modifications by a 32P-postlabeling assay, characteristic tissue-specific patterns of 32P-labeled spots (termed I-spots) were observed on thin-layer chromatograms. Amounts of these DNA derivatives (termed I-compounds), which were not detected in newborn rat DNA, markedly increased with age. This novel type of DNA modification could be due to environmental (e.g. dietary) factors or to endogenous DNA-reactive metabolites and may conceivably play a role in the initiation of spontaneous cancers or other adverse health effects related to aging.