The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH(2)-terminal domain (residues 1-140) within HSV-1 Pol. Due to the conservation of the pre-NH(2)-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5'-3' polymerase activity. We identified three pre-NH(2)-terminal Pol mutants that exhibited 5'-3' polymerase activity indistinguishable from that of wild type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA(6), in which a conserved motif at residues 44-49 was substituted with alanines. We constructed the corresponding pol mutant viruses and found that polΔN43 displayed replication kinetics similar to those of wild type virus, while polΔN52 and polA(6) infection resulted in an 8-fold defect in viral yield when compared to wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA(6) viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH(2)-terminal domain is important for viral DNA synthesis and production of infectious virus, and indicate a functional role for this domain.