The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.