A vaccinia-directed deoxyribonucleic acid (DNA) polymerase has been partially purified from the cytoplasmic fractions of virus-infected HeLa cells. The utilization of natural and synthetic templates by this enzyme resembles that of the host cell DNA-dependent DNA polymerases. The vaccinia DNA polymerase cannot copy ribopolymers or ribonucleic acid but is very effective with an "activated" DNA as template. An exonuclease preferring single-stranded DNA as substrate is found in the most highly purified preparations of the enzyme. The molecular weight of the vaccinia DNA polymerase seems to be about 110,000. The viral DNA polymerase is also found to be associated with purified, infected cell nuclei, and this association may be due, at least in part, to nonspecific adsorption of the vaccinia DNA polymerase by nuclei.