To accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct natural 2'-deoxy-5'-triphosphate (dNTP) opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs but does not require N-3 for correct polymerization. A free pair of electrons on N-1 also helps prevent misincorporation opposite A, C, and G and strongly drives polymerization opposite T. N6 contributes a small amount both for preventing misincorporation and for correct polymerization. Within the context of guanine in either the incoming dNTP or the template base being replicated, N2 prevents misincorporation opposite adenine but plays at most a minor role for incorporation opposite C. In contrast, adding N2 to the dNTPs of either adenine, purine, 6-chloropurine, or 1-deazapurine greatly enhances incorporation opposite C, likely via the formation of a hydrogen bond between N2 of the purine and O2 of the pyrimidine. Herpes polymerase is very sensitive to the structure of the base pair at the primer 3'-terminus since eliminating N-1, N-3, or N6 from a purine nucleotide at the primer 3'-terminus interfered with polymerization of the next two dNTPs. The biological and evolutionary implications of these data are discussed.