We report the characterization of a DNA primase/ polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus-and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases. PolpTN2 exhibited primase, polymerase and nucleotidyl transferase activities and specifically incorporates dNTPs, to the exclusion of rNTPs. PolpTN2 could efficiently prime DNA synthesis by the T. nautilus PolB DNA polymerase, suggesting that it is used in vivo as a primase for pTN2 plasmid replication. The N-terminal PriS-like domain of PolpTN2 exhibited all activities of the full-length enzyme but was much less efficient in priming cellular DNA polymerases. Surprisingly, the N-terminal domain possesses reverse transcriptase activity. We speculate that this activity could reflect an ancestral function of AEP proteins in the transition from the RNA to the DNA world.