Inhibition of varicella-zoster virus (VZV) replication by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been examined in vitro under various experimental conditions. The 50% inhibitory dose (ID50) of BVDU for VZV replication in human embryonal fibroblast (HEF) cultures was 0.016 micrograms/ml, if the assay was based on the reduction of visible foci. If the assay was based on the reduction of immunofluorescent foci, the ID50 was 3 times higher. There was no significant difference in ID50, whether the HEF cultures were infected with cell-free or cell-associated VZV. When the HEF cells were infected with VZV at different multiplicities of infection (MOI), the ID50 of BVDU increased in parallel with the increase of MOI. BVDU was normally added immediately after virus infection. However, the addition of BVDU could be delayed until 8 hr after infection without substantial decrease of activity. On the other hand, BVDU did not cause any inhibition of focus formation when it was removed within 8 hr after VZV infection. If removed at 24 or 48 hr after infection, BVDU caused a significant reduction in focus formation. BVDU had no effect on focus formation if added to the HEF cells before virus infection. BVDU was also found to inhibit VZV focus formation in Vero cells, but only at an ID50 that was 5-10 times higher than the ID50 noted in HEF cells.