This chapter explores how animal mitochondrial DNA (mtDNA) is expressed and replicated. The mtDNA contain only one significant stretch of nucleotide sequence that does not code for RNA or a protein molecule. This region is the displacement-loop (Dloop) portion, which is a signature feature of vertebrate mtDNA. The essential major cis-acting elements necessary for transcriptional initiation are located within the confines of the D-loop region, which is defined explicitly as that sequence bounded by the genes for transfer RNA (tRNA)-phenylalanine and tRNA-proline. The D-loop region also contains all of the required template information necessary for the initiation of nascent heavy (H)-strand DNA synthesis, which marks the beginning of a round of DNA replication in this system. Human mtDNA promoters include a short region encompassing the transcriptional start sites that has important sequence requirements, as evidenced by the fact that mutations in these start sites have serious consequences for promoter function. Efficient transcription requires the presence and action of the only identified trans-acting factor in vertebrate mitochondrial transcription. This factor, termed “mtTFl,” functions by binding immediately upstream of the transcriptional start sites from positions –10 to –40 at each promoter.