The posterior segment of the eye was divided into four compartments: retinal cytosol (R), subretinal fluid on the retinal surface (S/R), retinal pigment epithelial (RPE) cytosol, and subretinal fluid on the RPE surface (S/RPE). The volume of each compartment was estimated from the dilution of creatinine (in the extraction buffer) by the endogenous tissue fluid.The ascorbate concentrations in R, S/R, S/RPE, and RPE were 20.6, 12.3, 3.7, and 5.8 mg/dl respectively. Dehydroascorbate was observed only in the RPE and S/RPE. The decreasing ascorbate concentration from the retina to RPE, and the distribution of dehydroascorbate suggest a movement of ascorbate from the vitreous cavity into the subretinal space.The permeability of retinal cell layers to ascorbate was confirmed by the high radioactivity observed in the subretinal space after an intravitreal injection of C14-ascorbate. The occurrence of dehydroascorbate in the RPE and the S/RPE indicates the presence of oxidative reaction of ascorbate in these compartments, where light induced free radicals are located.