The displacement loop region of human mitochondrial DNA contains the origin of heavy strand DNA replication and is the most likely site of promotion of transcription of both heavy and light strands. In order to identify relevant control regions for initiation of transcription, a partially purified human mitochondrial RNA polymerase activity was isolated and utilized in a runoff transcription assay using a cloned portion of the displacement loop region as the DNA template. Analysis of the transcription products from differentially cleaved DNA templates reveals that specific light strand transcripts are synthesized and no heavy strand transcripts are detectable. The 5' ends of the light strand transcripts map within a unique trinucleotide site on the heavy strand template at a position which overlaps the pentanucleotide map position of the 5' ends of in vivo 7 S RNA light strand transcripts. By using templates that have been truncated at the 5' or 3' end, an upper limit on the size of template sequence required for synthesis of the specific light sequence required for synthesis of the specific light strand transcripts can be defined as the 433-nucleotide genomic region between the 5' 10 nucleotides of the 12 S rRNA gene and a BalI restriction site in the displacement loop region that is 352 nucleotides from the gene boundary for tRNAPhe. Two of the previously identified conserved sequences of the mammalian displacement loop region are not required for synthesis of the light strand transcripts. The location of the in vitro light strand transcripts is consistent with a functional role in either in vivo transcription or priming of heavy strand DNA replication.