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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.