Individual promoters for transcription of each strand of human mtDNA are located near the origin of heavy-strand DNA replication in the displacement-loop region. Initiation of heavy-strand synthesis represents the first event in mtDNA replication. Analyses of the 5' and 3' map positions of displacement-loop nucleic acids from mitochondria of cultured human cells reveal a close correspondence between the 3' ends of RNA, whose 5' ends map at a unique site, and the 5' ends of DNA strands. The 5' ends of the RNA species all map at nucleotide position 407 in the genomic sequence, which corresponds exactly to the major 5' transcriptional start site, determined previously in vitro, that is contained within the light-strand promoter sequence. Displacement-loop heavy-strand DNAs map immediately adjacent to the 3' termini of these RNAs, and these transition points between RNA and DNA lie within short conserved sequence blocks in the template sequence. The simplest interpretation of these data is that replication is initiated at the major transcriptional promoter with subsequent precise cleavage of primary transcripts to provide the appropriate primer species.