The transcription of DNA and processing of RNA in mitochondria was investigated using isolated HeLa cell mitochondria. The intact organelles transcribe their DNA and process their RNA both qualitatively and quantitatively similar to the in vivo situation. Changing the conditions for the transcription reactions allowed the identification of a processing pathway for the small ribosomal RNA species, and an RNA species from the region surrounding the origin of light-strand replication with novel electrophoretic properties. Removal of the mitochondria from the cellular environment simplified the investigations of the nuclear-cytoplasmic influences upon the mitochondrial transcription. Differential sensitivities of all three transcription events, synthesizing the rRNAs, mRNAs, and light-strand RNAs, was shown to exist to a small molecular weight factor(s) present in the cytoplasm, and the availability of energetic substrates. These sensitivities indicate a link between cytoplasmic and respiratory/oxidative phosphorylative control of mitochondrial transcription.