The non-coding region of rabbit mitochondrial DNA (mtDNA) exhibits two sets of tandem repeats between conserved sequence block 1 (CSB1) and the tRNA Phe gene. Both repeated sequences, short repeated (SR) and long repeats (LR), which contain 20 and 153 nucleotides, respectively, are involved in the generation of a high degree of mitochondrial heteroplasmy. Due to the location of these sequences in the regulatory region and their properties in terms of variable conformations, they could affect the initiation of replication of the heavy-strand DNA (H-strand DNA) and subsequently would influence the efficiency of mtDNA replication. The extremities of the displacement loop (D-loop) DNA strand and the 5' ends of RNA primers initiating the H-strand DNA synthesis were characterized in individual rabbits. Mapping at the nucleotide level of the 5' and 3' ends of the D-loop DNA strands indicates that both extremities are heterogeneous. The H-strand replication origin OH is located close to the conserved sequence block CSB1 as in other mammals. In all of the individuals studied so far, DNA molecules with a 5' end 1-2 nucleotides downstream of CSB1 were always present. As H-strand DNA replication is believed to be primed by RNA transcribed from the light-strand promoter (LSP), RNA mapping was carried out to identify the 5' end of H-strand RNA. Neighbouring initiation sites were identified at the nucleotide level in an (A+T)-rich region at nucleotide 1841 and in a stretch of cytosine residues at nucleotides 1849-1852, which are located at the beginning of the first long repeat. A detailed RNA analysis indicates that H-strand RNA molecules are initiated in each long repeat. The amplification of the regulatory region has produced multiple initiation transcription sites and a family of RNA primers of various lengths. These variations in length and the ensuing secondary structures are not critical for their potential function as H-strand DNA replication primers.