The conserved nonanucleotide sequence functions as a promoter in the yeast (Saccharomyces cerevisiae) mitochondrial genome. A mitochondrial gene, Oli 1, which codes for ATPase subunit 9, has two identical nonanucleotide promoter sequences separated by 78 nucleotides, but they initiate transcription with very different efficiencies in vivo and in vitro. Deletion analysis has revealed that the nucleotide at position +2 of the weak downstream promoter accounts for its poor in vitro transcriptional activity. This finding was confirmed with site-specific mutations at +2 and +3 positions of a consensus synthetic promoter. The nonanucleotide mitochondrial promoter with a pyrimidine at position +2 acts as a weak promoter, whereas the same sequence with a purine at the +2 position functions as a strong promoter. The nucleotide at the +3 position further contributes to the relative promoter strength. These results suggest not only that the conserved nine-nucleotide sequence is required for the correct transcriptional initiation but also that other neighboring nucleotides influence the efficiency of promoter function.