The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.