Rat liver mitochondrial (mt) DNA was investigated for the presence of I-compounds, a recently discovered type of DNA modifications which is detected and measured via 32P-postlabeling. These DNA modifications were previously shown to accumulate in an age-dependent manner in total cellular DNA of various tissues of untreated rodents. In the present work, mt DNA of 1-, 3-, 6-, and 9-month-old female Sprague-Dawley rats was found by 32P-postlabeling also to contain I-compounds that increase with age. Most of the I-compounds were identical for mt and nuclear (nu) DNA. A cluster of 2 non-polar I-spots (termed M-compounds) was mitochondria-specific and increased about 8-fold from 1 to 9 months, attaining a RAL value of 44 X 10(-9) or 1 modification in 2.3 X 10(7) DNA nucleotides at 9 months. Quantitative differences between chromatographically identical spots were seen mainly for a low-polarity fraction of I-compounds, which exhibited 2 times higher overall levels in mt DNA versus nu DNA over the age range studied. Total I-compound levels increased during this time 6.9- and 5.1-fold in nuclei and mitochondria, respectively. The M-compound level was close to 10% of total mt DNA I-compound levels. M-compounds may conceivably be derived from potentially DNA-reactive electron carriers of the mt electron-transport chain, while I-compounds common to both mt and nu DNA presumably originate in extramitochondrial sources. The similarity of mitochondrial and nuclear I-compound profiles and amounts implies possible regulatory mechanisms in I-compound formation and repair. Mt DNA maps showed additional 32P-labeled material which may have been associated with DNA damage caused by oxygen free radicals known to be generated by the mt electron-transport chain. Age-dependent increases of mt DNA modifications are potentially related to mt mutations and may be linked to age-related degenerative changes in mitochondria.