PURPOSE: To delineate the vitreal pharmacokinetics of dipeptide monoester prodrugs of ganciclovir (GCV) with conscious rabbit model using ocular microdialysis and to compare with published results from anesthetized model. METHODS: New Zealand albino male rabbit was selected as the animal model. Conscious animal ocular microdialysis technique with permanently implanted probes was employed to delineate the pharmacokinetics of GCV, L-valine-GCV (Val-GCV), and dipeptide monoester GCV prodrugs [val-val and L-glycine-val (Gly-Val)] after intravitreal administration. RESULTS: This work employs conscious model to evaluate vitreal pharmacokinetic parameters and compares the results with previously published data from anesthetized animal, thereby demonstrating the effect of anesthesia on the vitreal disposition of dipeptide prodrugs of GCV. Results have revealed that area under curve (AUC), clearance, and last measured plasma concentration (C(last)) for all 4 compounds were significantly altered in a conscious animal relative to the anesthetized model, while mean residence time (MRT) was significantly reduced. However, the AUCs of regenerated Val-GCV and GCV from Gly-Val-GCV and Val-Val-GCV were found to be unchanged, suggesting higher ocular metabolism in conscious animals. CONCLUSION: This study for the first time delineates the vitreal pharmacokinetics of a GCV prodrug in conscious animals and compares the data with anesthetized animals. Lower vitreal exposure levels were obtained in case of conscious animal model; however, the elimination rates were not influenced by anesthesia.