The purpose of this study was to determine the pharmacokinetics governing the distribution and elimination of intravitreally injected vancomycin in normal and infected rabbit eyes. Two groups each of 36 pigmented animals were used. Group 1 served as control. In Group 2, experimental endophthalmitis was induced in the right vitreous by inoculation with Staphylococcus aureus. Once endophthalmitis developed, a vancomycin solution was injected. Four animals from each group were killed at nine time points post-injection, the vitreous and aqueous were removed, and blood samples were taken for HPLC analysis. Data analysis was performed using the RSTRIP program. The half-lives were 69 hours in normal vitreous and 14.53 hours in infected vitreous. Therapeutic drug levels were present in the vitreous 84 hours post-injection in all eyes; they were detected from 2 to 48 hours in normal aqueous but at lower levels in the infected ones. Kv and Ca/Cv ratios suggested that the primary route of elimination was across the retina and the anterior chamber in normal eyes, and via the retina in infected eyes. Results indicate that pharmacokinetic parameters change in pathological conditions, which may help establish better treatment guidelines for endophthalmitis.