Yeast mitochondrial DNA contains multiple promoters that are responsible for expression of its genes. The basic yeast mitochondrial promoter consists of a nonanucleotide consensus sequence [5'-ATATAAGTA(+1)-3'] that must be recognized by transcription proteins, including mitochondrial RNA polymerase and any relevant trans-acting factors. Since mitochondrial RNA polymerase alone appeared unable to recognize a mitochondrial promoter, we examined the effects of providing accessory proteins to enable promoter function. After expression in Escherichia coli or purification from yeast mitochondria, two proteins were tested; they were ABF2 (a structural homologue of the human mitochondrial transcriptional activator mtTF1) and MTF1 (the gene product of a yeast locus known to exhibit a mitochondrial transcription phenotype). The results show that MTF1 specifies correct transcriptional initiation while ABF2 does not. We conclude that MTF1 is an essential key protein in yeast mitochondrial promoter function. Considering the increasing complexity of the mitochondrial transcription apparatus, we propose a nomenclature system for its components.