Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A. Both TFB1M and TFB2M interact directly with POLRMT, but TFB2M is at least one order of magnitude more active in promoting transcription than TFB1M. Both factors are highly homologous to bacterial rRNA dimethyltransferases, which suggests that an RNA-modifying enzyme has been recruited during evolution to function as a mitochondrial transcription factor. The presence of two proteins that interact with mammalian POLRMT may allow flexible regulation of mtDNA gene expression in response to the complex physiological demands of mammalian metabolism.