Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100-200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.