A discrete primary transcript of HeLa mitochondrial DNA containing 12 S rRNA, tRNAPhe, and 19 +/- 2 nucleotides upstream has been identified. The 5' ends of heavy strand encoded RNAs adjacent to the D-loop region of mitochondrial DNA were mapped by primer extension and S1 nuclease protection to a site 19 +/- 2 nucleotides upstream from the start of tRNAPhe. Hybridization of a tRNAPhe-specific probe to a blot of mitochondrial RNA detected a discrete species with a molecular weight consistent with an RNA containing 12 S rRNA, tRNAPhe, and a short upstream leader sequence. The di- or triphosphate nature of the 5' end of this polycistronic RNA was demonstrated by in vitro capping with [alpha-32P]GTP and guanylyltransferase. S1 nuclease protection of the purified in vitro capped RNA established that the 5' end, 19 +/- 2 nucleotides upstream from tRNAPhe, is an initiation site for heavy strand transcription.