Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.