Plasmids containing the origin region of human mtDNA are specifically transcribed by the partially purified homologous mtRNA polymerase in vitro. Transcription of both the light and heavy strands initiates at the same sites previously identified as in vivo transcription start sites. The sequences responsible for initiation of heavy strand transcription are investigated in detail, since this transcript includes the rRNA cistrons and the majority of tRNA genes and potential mRNAs. Deletion mutagenesis is employed to delimit the sequences responsible for heavy strand transcription to a region of less than 35 bp surrounding the transcription start site. This region contains a repetitive sequence, AAACCCC, and shows homology to other regions of the human mtDNA that have been implicated as transcription initiation sites or that show unusual homology to other mammalian mtDNA genomes.