Isolated rat brain mitochondria, when incubated in the presence of [alpha-32P]UTP in an appropriate incubation buffer, in which the energy requirements are provided by exogenous ADP in the presence of an oxidizable substrate, are able to support mitochondrial DNA transcription and RNA processing in a way faithfully resembling the in vivo process. Furthermore, we have strikingly found a saturation of the synthesis of mature 16 S and 12 S rRNA under conditions in which their RNA precursor as well as the mature mRNAs continue being synthesized. This suggests that synthesis of mature rRNAs could be regulated at the level of processing of their precursor rather than at the level of transcription.