The yeast mitochondrial RNA polymerase (RNAP) is a two-subunit enzyme composed of a catalytic core (Rpo41) and a specificity factor (Mtf1) encoded by nuclear genes. Neither subunit on its own interacts with promoter DNA, but the combined holo-RNAP recognizes and selectively initiates from promoters related to the consensus sequence ATATAAGTA. To pursue the question of why Rpo41, which resembles the single polypeptide RNAPs from bacteriophage T7 and T3, requires a separate specificity factor, we analyzed a collection of Mtf1 point mutations that confer an in vivo petite phenotype. These mutant proteins are able to interact with Rpo41 and are capable of nearly wild type levels of initiation in vitro with a consensus promoter-containing template (14 S rRNA). However, the petite phenotype of two mutants can be explained by the fact that they exhibit dramatic transcriptional defects on non-consensus promoters. Y54F is incapable of transcribing the weak tRNA(Cys) promoter, and C192F cannot transcribe either tRNA(Cys) or the variant COX2 promoter from linear DNA templates. Transcription of the tRNA(Cys) promoter by both mutants was significantly corrected by addition of an initiating dinucleotide primer or by supercoiling the DNA template. These results establish the critical role of Mtf1 in promoter recognition and initiation of transcription.