E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) and E-5-(2-iodovinyl)-2'-deoxyuridine (IvdUrd) are among the most potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) replication. To elucidate the site of inhibition, we examined whether the halovinyl analogs are incorporated into DNA using two approaches. (i) In assays with purified DNA polymerases omitting dTTP from the reaction system, addition of either BrvdUTP or IvdUTP increased the polymerization reaction, indicating that these two analog triphosphates can be alternate substrates. (ii) When HSV-1-infected Vero cells were grown in the presence of either BrvdUrd or IvdUrd, there was an increase in the density of both the viral and cellular DNA. The viral DNA had 40% of its thymidine moiety substituted by IvdUrd when the concentration of [125I]IvdUrd was 24 microM (in the absence of added thymidine). At 30 microM BrvdUrd and 1 microM [2-14C]thymidine, the viral DNA had only 11% of its thymidine moiety substituted by BrvdUrd, presumably because of the presence of added thymidine. Following digestion of [125I]IvdUrd-substituted DNA with DNase 1, venom phosphodiesterase, and alkaline phosphatase, the radioactivity co-migrated with nonradioactive IvdUrd in thin layer chromatography. Under similar conditions, no detectable incorporation of either [125I]IvdUrd or BrvdUrd into mock-infected Vero cell DNA was observed. Thus, IvdUrd and BrvdUrd are incorporated into DNA of HSV-1 infected cells but not into DNA of uninfected cells.