We investigated mutations within the presumed 3'-5' exonuclease domain of the DNA polymerase from herpes simplex virus type 1. The mutation sites correspond to residues in DNA polymerase I (Escherichia coli) which bind two metal ions that are required for exonuclease function. To evaluate the effect of the herpesvirus mutations on enzymatic activity, we overexpressed the wild-type DNA polymerase and one mutant enzyme using a baculovirus expression system. Both proteins exhibited DNA polymerase activity after partial purification, but the mutant protein was drastically deficient in exonuclease activity. This finding suggests that the herpesvirus exonuclease may utilize the same metal-ion-mediated mechanism employed by DNA polymerase I. We also attempted to transfer each of the mutations into the herpesvirus genome using a marker rescue protocol. Although wild-type sequences could be transferred readily, recombinant viruses carrying mutant sequences were not recovered. We discuss the possibility that the mutations are lethal and suggest mechanisms by which a deficiency in 3'-5' exonuclease might cause loss of viability.