Critical elements of a mammalian mitochondrial DNA heavy-strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI). We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; hybrid formation was dependent on the conserved CSBII element. We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA hybrid formation is also evident at the human mitochondrial heavy-strand origin. As predicted, hybrid formation was dependent on the GC-rich CSBII element. The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication. Interestingly, efficient hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA hybrid, including the CSBIII element. These results suggest that the RNA-DNA hybrids formed during transcription across the mitochondrial DNA heavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication.