The effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on herpes simplex virus (HSV) replication was examined and compared with that of E-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd). The 50% inhibitory dose against HSV type 1 (HSV-1) was 0.1 microgram/ml compared with 0.008 microgram/ml for BVdUrd; the antimetabolic 50% inhibitory dose of BVaraU ranged from 20 to 95 micrograms/ml. The addition of 50 micrograms of BVaraU per ml to HSV-1-infected Vero cells decreased the synthesis of viral and cellular DNA by 37 and 28%, respectively. The 5'-triphosphate (BVaraUTP) competed with dTTP in DNA synthesis by the herpes-viral and cellular DNA polymerases; the apparent Ki values of HSV-1 DNA polymerase, DNA polymerase alpha, and DNA polymerase beta were 0.14, 0.32, and 5 microM, respectively. Thus, BVaraU was a less effective antiherpesvirus agent than BVdUrd; unlike BVdUrd, it did not appear to be internally incorporated into replicating DNA in virus-infected cells.