We have purified the protein that confers selective promoter recognition on the core subunit of the yeast mitochondrial RNA polymerase. The N-terminal sequence of the 43-kDa specificity factor identified it as the product of the MTF1 gene described by Lisowsky and Michaelis (1988). We confirmed that MTF1 encoded the specificity factor by analyzing extracts from a yeast strain bearing a disruption of the gene. The extracts contained normal levels of core RNA polymerase but lacked selective transcription activity; adding the purified 43-kDa protein restored selective transcription. Comparison of the MTF1 protein sequence to the family of bacterial sigma factors has revealed striking similarity to domains identified with--10 promoter recognition, promoter melting, and holoenzyme stability.