Thymidine kinase-deficient mouse cell cultures infected with herpes simplex virus type 1 exhibited a maximum of virus DNA synthesis around 8 h post-infection as determined by pulse labelling with 3H-thymidine. Cellular DNA synthesis was progressively inhibited, but still appreciable until 8 h post-infection and not completely abolished at any time during the infectious cycle. Phosphonoacetic acid was found to be a potent and selective inhibitor of virus DNA synthesis only when added to infected cultures before the onset of virus DNA synthesis. During the interval of increasing virus DNA synthesis the activity of cellular alpha polymerase decreased rapidly, whereas the beta polymerase activity increased significantly; a slight increase was observed for the gamma polymerase activity. When infected cells were kept in the presence of phosphonoacetic acid following virus adsorption the effect on cellular DNA polymerases was less pronounced.