Peptides from preselected regions of the herpes simplex virus DNA polymerase were used to generate monospecific antisera to defined regions of the enzyme. The antisera were used to localize the polymerase within the infected cell and to determine the time of synthesis during productive infection. Comparison with a neutralizing polyclonal antiserum was used to show the specificity of the peptide antisera. By using the antisera the stabilities of the DNA polymerase, the alkaline nuclease, and the major DNA-binding protein were determined, and the state of phosphorylation of the DNA polymerase was compared with each of these proteins.