The two major promoters for transcription of the human mitochondrial genome are located near each other in the displacement-loop region of the molecule. Previous work has localized these promoters to regions of < 100 nucleotides each; the DNA sequence at the transcription start site is stringently required, as is the region from −10 to −40 base pairs upstream of each respective start site. Each upstream site is recognized and bound by human mitochondrial transcription factor A (h-mtTFA), an event previously shown to be important for transcriptional activation. We report here results using recombinant h-mtTFA that demonstrate the dependence of transcription initiation on h-mtTFA. In addition, altering the distance between the h-mtTFA binding site and the transcription start site greatly impairs transcription initiation efficiency. The decrease in transcription initiation efficiency was shown to be a consequence of altering the position of h-mtTFA binding as opposed to the strength of h-mtTFA binding, as judged by DNA footprinting ability. Analysis of a chimeric yeast-human promoter revealed that the yeast mtTFA homologue cannot substitute for the human protein, even when bound at an appropriate position upstream of the human transcription start site.