We report the identification of a family of four active genes (Ehodp1, Ehodp2, Ehodp3, and Ehodp4) encoding putative DNA polymerases in Entamoeba histolytica, the protozoan parasite responsible of human amoebiasis. The four Ehodp genes show similarity to DNA polymerases encoded in fungi and plant mitochondrial plasmids. EhODP polypeptides conserve the 3'-5' exonuclease II and 5'-3' polymerization domains, and they have the I, II, and III conserved boxes that characterize them as DNA polymerases of family B. Furthermore, we found in EhODP polymerases two novel A and B boxes, present also in DNA polymerases encoded in fungi mitochondrial plasmids. By in situ PCR, Ehodp1 gene was located in nuclei and in DNA-containing cytoplasmic structures. Additionally, using polyclonal antibodies against a recombinant rEhODP1-168 polypeptide, and confocal microscopy, EhODP1 was located in cytoplasmic DNA-containing structures.