The herpes simplex virus DNA polymerase provides an excellent model for studies of eukaryotic replicative polymerases. We report here the nucleotide sequence of the gene which encodes this enzyme. The gene includes a 3705-base-pair major open reading frame capable of encoding a Mr 136,519 polypeptide, in rough agreement with previous estimates of the size of the major polypeptide found in partially purified viral polymerase preparations. The predicted polymerase polypeptide shares extensive sequence homology with the Epstein-Barr virus open frame predicted to encode DNA polymerase and with a 13-amino acid segment of adenovirus 2 DNA polymerase. Mutations conferring altered sensitivity to antiviral deoxynucleoside triphosphate analogs, pyrophosphate analogs, or aphidicolin from eight different mutants map within the region encoding the carboxyl-terminal portion of the predicted polymerase polypeptide. Two of these are separated by a distance corresponding to at least 228 amino acids. We propose that this region of the gene encodes a polypeptide domain that contains the binding sites for deoxynucleoside triphosphates and pyrophosphate.