The content of DNA and of 16S rRNA and of two mRNAs, i.e., the mRNA for the cytochrome c oxidase subunit I and the mRNA for one subunit of the NADH dehydrogenase (ND4), in free (nonsynaptic) mitochondria of developing and adult rat cerebellum has been determined. During postnatal development, DNA content of free (nonsynaptic) mitochondria increases 10 times from 1 to 30 days of age whereas, in adult rats, it is about 60% compared to that found in 30-day-old rats. The total content of each RNA species studied also increases during development. However, when the content of each RNA is expressed per mtDNA molecule, rRNAs and mRNAs behave differently: 16S rRNA level does not change during development and it is not significantly different from that of the adult rat, whereas the level of mRNAs is higher during development than in the adult rat and changes with age. These results are discussed in light of mitochondrial biogenesis in rat cerebellum during development and of the regulation of the mitochondrial DNA transcription process.