This chapter describes a system to study RNA synthesis by using isolated mitochondria from mammalian brain and liver. In this system, mtDNA transcription and RNA processing are supported in a way that closely reproduces the vivo process. In vivo study of the regulation of mtDNA expression is hampered by the inherent difficulty of manipulating mammalian mitochondrial genes in vivo and of discriminating between direct regulatory phenomena on mtDNA transcription and RNA metabolism and secondary actions dependent on nuclear control. Based on this experimental approach, several attempts are made to apply this system to the study of mitochondrial RNA synthesis in mammalian organs. However, under the incubation conditions described for the organelles isolated from HeLa cells, the mitochondria isolated from the mammalian tissue do not reproduce closely the in vivo processes of mtDNA transcription and RNA processing and the efficiency of transcription is very low.