The major site of in vivo transcriptional initiation for both heavy and light strands of human mitochondrial DNA is the displacement-loop region. Transcripts synthesized in vitro by human mitochondrial RNA polymerase were mapped to the nucleotide level and have identical 5' end map positions to those reported for in vivo primary transcripts. An ordered series of deletion clones, whose template sequences were truncated at either the 5' or 3' end, was used to identify the precise mitochondrial DNA sequence required for initiation of transcription. The data provide a definitive assignment of the promoter for heavy-strand transcription occurring within -16 to +7 of the transcriptional start site 16 nucleotides upstream of the 5' end of the gene for tRNAPhe and of the promoter for light-strand transcription occurring within -28 to +16 of the transcriptional start site at the 5' end of "7S RNA." Within each control sequence is a candidate promoter whose consensus sequence is 5'-CANACC(G)CC(A)AAAGAPyA-3' and in both cases transcriptional initiation occurs within six to eight nucleotides of the 3' end of this sequence. The transcriptional start site is an integral part of each promoter and each promoter can function in the absence of the other.