DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.