The physical isolation of mammalian mitochondrial DNA (mtDNA) over 30 years ago marked the beginning of studies of its structure, replication and the expression of its genetic content. Such analyses have revealed a number of surprises: novel DNA structural features of the circular genome such as the displacement loop (D-loop); multiple sized and deleted forms of the circular genome; a minimal set of mitochondrially encoded rRNAs and tRNAs needed for translation; a bacteriophagelike, nuclear-encoded mitochondrial RNA polymerase for transcription; and a direct linkage between transcription and the commitment to replication of the leading mtDNA strand that centres on the nuclear encoded mitochondrial transcription factor A. One of the more recent revelations is the existence, near the D-loop, of an atypical, stable RNA-DNA hybrid (or R-loop) at the origin of mammalian leading-strand DNA replication, composed of the parent DNA strands and an RNA transcript. In mammalian mitochondrial systems, all of the proteins known to be involved in DNA replication are encoded in the nucleus. Thus alterations and deficiencies in mtDNA replication must arise from mutations in mtDNA regulatory sequences and nuclear gene defects. Further studies of the relationships between nuclear-encoded proteins and their mtDNA target sequences could result in strategies to manipulate genotypes within cellular mtDNA populations.