In order to understand the cause of the reduced mitochondrial DNA transcription in heart and brain of senescent rat previously reported, we focused our attention on the content and structure of rat mitochondrial DNA in adult and senescent rats. The estimate of the mtDNA copy number in liver, heart and brain of adult and senescent rats showed that in all organs examined the senescent individuals have a mtDNA content higher than the adult counterparts. The analysis of mtDNA structural changes involved the search for point mutations and large deletions. As for the first case, the determination of the nucleotide sequence of many independent clones containing two mtDNA restriction fragments isolated from rat cerebral hemispheres did not show any sequence difference between adult and senescent individuals. However, analysis of mtDNA deletions by the polymerase chain reaction in liver and brain of adult and senescent rats identified a small population of mtDNA molecules harboring a deletion of 4834 bp. The estimate of the proportion of deleted molecules in the liver showed that they represent 0.02% and 0.0005% of total mtDNA in senescent and adult rat liver respectively. Therefore, a mtDNA deletion also accumulates in the rat during aging. This result supports the hypothesis of the accumulation of deleted mtDNA molecules in aging. However, the low percentage of deleted mtDNA molecules already found and the reversibility of the reduced mitochondrial DNA transcription in senescent rat raise doubts on the primary role of the irreversibly damaged mtDNA molecules in aging. Deleted mtDNA molecules along with changes caused by lipid peroxidation of mitochondrial membranes might contribute to the overall decline of mitochondrial function.