Mitochondrial promoters in Saccharomyces cerevisiae contain an identical octanucleotide [sequence: see text] sequence present just upstream of the initiation site (at the left end of the arrow). Studies have shown that the transcription rates of mitochondrial genes vary from 7- to 15-fold. The nucleotide at position +2 regulates the efficiency of mitochondrial promoters but does not affect the specificity of initiation. The data presented herein demonstrate that the variable transcription rates of mitochondrial genes are due to different levels of transcriptional initiation. The rate of first phosphodiester bond formation between a purine and a pyrimidine on a weak promoter is much lower than that of purine-purine on a strong promoter. A dinucleotide corresponding to positions +1 and +2 acts in vitro as a primer, bypassing the first phosphodiester bond formation at the initiation site. When these dinucleotides were used to prime transcription, the activities of the strong and weak promoters were found to be identical. In heparin-challenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation. These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.