Purpose.To study the effects of antiviral agents on human keratocytes in vitro.Methods.Cultured human keratocytes were incubated with either ganciclovir, idoxuridine, trifluridine, or cidofovir at concentrations from 0.0001 to 10 mg/mL. Phase-contrast microscopy and XTT (sodium [2,3-bis [2-methoxy-4-nitro-5-sulphophenyl]-2h-tetrazolium-5-carboxanilide, inner salt) colorimetric assay were performed after 24, 48, and 72 hours of incubation.Results.When adjustments were made for time of incubation and concentration, trifluridine reduced cell viability significantly more than ganciclovir, idoxuridine, and cidofovir (p< 0.001, three-way analysis of variance). There was significant time-and dose-dependent reduction of cell viability (p< 0.001) with trifluridine and cidofovir. After a 72-hour incubation with ganciclovir or idoxuridine, cell viability was reduced as compared with 24-and 48-hour incubation (p< 0.001); only the effects of the highest concentration tested (1.0 mg/mL) were significantly different from those of the lower concentrations (p< 0.002). At a concentration of 1.0 mg/mL, trifluridine and cidofovir produced moderate to severe signs of cytotoxicity, whereas ganciclovir and idoxuridine displayed much less severe morphologic signs.Conclusions.Our results indicate that antiviral agents may have both time-and concentration-related toxic effects on stromal keratocytes. These findings may impact the selection of the most appropriate antiviral drug when it is needed to treat infections involving the corneal stroma.