The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.