Vitreous fluorophotometry assesses the leakage of fluorescein through the primary blood-retinal barrier composed of retinal capillaries and the pigment epithelium. However, other ocular barriers exist that may differentiate between the small molecules of fluorescein and the larger ones of protein. We injected solutions containing carboxyfluorescein and several sizes of fluoresceinated dextran into the subretinal space of rabbit eyes. Carboxyfluorescein diffused rapidly through the retina into the vitreous, but dextrans of a size comparable to proteins crossed the retina very poorly. Large dextrans persisted for days in the subretinal space, although the fluid injected with them disappeared within a few hours. Vitreous fluorophotometry must be interpreted with the knowledge that the retina is much more of a barrier to protein than to fluorescein.