During mitochondrial DNA (mtDNA) replication, DNA/RNA heteroduplex intermediates are formed. To understand how and why ribonucleotides are involved in mtDNA replication, we have studied the novel RNA-associated activities of human mitochondrial DNA polymerase (Pol gamma), including reverse transcription, RNA-directed 3' --> 5' DNA excision, RNA-primed DNA synthesis, and ribonucleotide incorporation. Remarkably, Pol gamma catalyzes reverse transcription with a slightly higher efficiency than HIV-1 reverse transcriptase, suggesting that the activity may be physiologically significant, and furthermore, proofreading activity with an RNA template was also observed. RNA-primed DNA synthesis activity is required for initiation of mtDNA replication, and we have found that Pol gamma holoenzyme is capable of performing this reaction at a physiologically relevant rate and that the accessory subunit plays an essential role in the initiation steps. Single ribonucleotides have been found scattered in the mtDNA genome, although their role and significance are not yet defined. Our finding that Pol gamma also incorporates ribonucleotide triphosphates into a DNA primer offers a plausible enzymatic pathway for the origin of the RNA-containing mtDNA genome.