PURPOSE: The aim of this study was to evaluate a series of dipeptide monoester ganciclovir (GCV) prodrugs with the goal of improving ocular bioavailability of GCV from topical ophthalmic solutions. METHODS: Solubility, logP, pH-stability profile, permeability, interaction with corneal peptide transporter, and in vivo efficacy against herpes simplex virus type 1 (HSV-1) ocular disease in the rabbit model were studied. RESULTS: Val-Val-GCV, Tyr-Val-GCV, and Gly-Val-GCV were more stable in aqueous solution than Val-GCV, showing no measurable degradation even after 7 d at 37 degrees C, within the pH range of 1.4-5.4. Tyr-Val-GCV and Val-Tyr-GCV were the most lipophilic among the prodrugs synthesized and were predicted to have an n-octanol/water partition coefficient 33 times greater than that of GCV. All of the prodrugs had a much higher aqueous solubility than the parent drug. Transcorneal permeability of Val-GCV and Val-Val-GCV was seven- to eightfold greater than that of GCV, in the presence of a proton gradient, and was significantly decreased in the presence of Gly-Pro. Val-Val-GCV (1% w/v) provided significantly better therapeutic activity than trifluorothymidine (1% w/v) against HSV-1 epithelial keratitis and equivalent therapeutic activity against stromal keratitis in the rabbit eye model. CONCLUSIONS: Val-Val-GCV demonstrates excellent corneal permeability and chemical stability, high aqueous solubility, and substantial in vivo antiviral activity against the HSV-1.