Combined effects of ganciclovir (GCV) and vidarabine (ara-A) on the replication, DNA synthesis, and gene expression of wild type-1 herpes simplex virus (HSV-1) and three acyclovir (ACV)-resistant HSV-1 mutants were studied. These mutants include a virus expressing no thymidine kinase (ACV^r), a virus expressing thymidine kinase with altered substrate specificity (IUdR^r), and a mutant expressing altered DNA polymerase (PAA^r5). GCV, an agent activated by herpesvirus specific thymidine kinase, showed potent antiviral activity against the wild type HSV-1(KOS) and DNA polymerase mutant (PAA^r5). The ACV-resistant mutants with thymidine kinase gene (ACV^r and IUdR^r) were resistant to GCV. All tested wild type HSV-1 or ACV-resistant HSV-1 mutants did not display resistance to vidarabine (are-A). Combined GCV and ara-A showed potentiating synergistic antiviral activity against wild type KOS and PAA^r5, and showed subadditive combnined ativiral activity against thymidine kinase mutants. Combined GCV and ara-A more significantly inhibited the viral DNA synthesis in wild type KOS and PAA^r5-infected cells to a greater extent than either agent alone, but the synergism was not determined in ACV^r or IUdR^r-infected cells. These data clearly indicate that combined GCV and ara-A therapy might be useful for the treatment of infections caused by wild type HSV-1 or ACV-resistant HSV-1 with DNA polymerase mutation. ACV-resistant viruses with the mutation in thymidine kinase gene are also, resistant to GCV, but susecptible to ara-A, indicating that ara-A would the drug of choice for the treatment of ACV-resistant HSV-1 which does not express thymidine kinase or expresses thymidine kinase with altered substrate specificity. While the synthesis of viral α-proteins of wild type HSV-1 was not affected by ACV, GCV, ara-A, or combined GCV and ara-A, the synthesis of β-proteins was slightly but significantly increased at the later stage of viral infection by the antiviral agents. The synthesis of γ-proteins of wild type HSV- 1 was significantly inhibited by ACV, GCV, ara-A, and combined GCV and ara-A. Combined GCV (5-μM) and ara-A (100-μM) also significantly altered the expression of viral β-and γ-proteins, of which efffct was similar to that of GCV (10-μM) alone. Although ACV at the concentration of 10-μM did not alter the expression of α-, β-, and γ-proteins of ACV-resistant PAA^r5, GCV and ara-A significantly alter the epression of β-and γ-proteins, not α-protein, as same manner as they altered the expression of those proteins in cells inffcted with wild type HSV-1. Combined GCV (5-μM) and ara-A (100-μM) altered the expression β-and γ-proteins in PAA^r5 infected cells, and the effect of combined regimen was comparable of that of GCV (10-μM). These data indicate that the alteration in the expression of β-and γ-proteins in wild type HSV-1 or PAA^r5 infected cells could be more significantly affected by combined GCV and are-A than individual GCV or ara-A.