The effects of ketoconazole alone and in combination with acyclovir and adenine arabinoside upon the replication of herpes simplex virus types 1 and 2 (HSV-1 and -2) were investigated by using a yield reduction assay. Ketoconazole demonstrated antiviral activity against HSV-1 and -2 and synergistic antiviral activity when it was combined with acyclovir. Combinations of ketoconazole with adenine arabinoside resulted in either interference or indifference. The effects of ketoconazole upon the protein synthesis of HSV-2-infected cells were also determined in an effort to define the mechanism of action for the antiviral activity of ketoconazole. There was no reduction of' HSV proteins when compared with acyclovir. These findings suggest that further investigations of the use of ketoconazole for the treatment of HSV infections are warranted.Recently, several reports have described a novel approach to antiviral chemotherapy with the antifungal' agent amphotericin B. Amphotericin B, a polyene antibiotic, exerts its antifungal effect by binding to sterol components in the fungal cell membrane, which results in increased permeability of the membrane (11). By using the water-soluble the combined effects of amphotericin B and acyclovir upon the replication of pseudorabies virus. Low doses of amphotericin B in combination with acyclovir produced a synergistic reduction in viral replication as shown with a plaque reduction assay. The exact mechanism for this synergistic effect was not elucidated (9).In view of this promising work, we investigated the possibility of antiviral activity for a different antifungal agent, ketoconazole. Ketoconazole is an imidazole compound which' exerts its antifungal activity through inhibition of lanosterol demethylation. This blocks the synthesis of ergosterol, the major sterol component of the fungal cell membrane (2). In mammalian cells, ketoconazole also inhibits lanosterol demethylation, with a subsequent decrease in the biosynthesis of cholesterol, the major sterol component of mammalian cell membranes. In addition, ketoconazole interferes with cellular fatty acid and phospholipid biosynthesis (2). In this study, we examined the effects of ketoconazole alone and in combination with acyclovir and adenine arabinoside upon herpes simplex virus types 1 and 2 (HSV-1 and -2) replication with a yield reduction assay. 107 PFU/ml were grown in monolayers of Vero cells and stored at -70°C.Cells. Vero and human lung (HL) cells (derived from an HL carcinoma) were used in all assays. Vero cells were grown in RPMI 1640 supplemented with 10% inactivated fetal bovine serum, 1% glutamine, 100 U of penicillin per'ml, and 50 jig of streptomycin per ml; and HL cells were grown in Hanks balanced salt solution, basal medium Eagle improved, supplemented with 10% inactivated fetal bovine serum, 1% glutamine, 100 U of penicillin per ml, and 50 ,ug of streptomycin per ml. Maintenance medium for both cell lines was supplemented with 1% inactivated fetal bovine serum in place of 10% inactivated fetal bovine serum.Dr...