A study was made on the permeability of blood vessels and connective tissue sheaths in the retina and optic nerve. Albumin labelled with Evan's blue (EBA), horseradish peroxidase (HP) or colloidal carbon were injected intravenously or into the orbit of mice, rats and rabbits. The tracers were then localized by light-, fluorescence-or electron microscopy. Blood vessels in retina and optic nerve were impermeable to all tracers. Intercellular diffusion between retinal endothelial cells of HP was prevented by tight junctions and its transcytoplasmic passage by pinocytosis across the endothelial lining was insignificant. Blood vessels in the optic nerve sheaths and in the soft tissues of the orbit differed from those inside the optic nerve in being permeable to the tracers (EBA and HP). Extracellular diffusion of the tracers from the orbit into the major portion of the optic nerve was prevented by a cellular lining covering the surface of the nerve. However, at lamina cribrosa the tracers could penetrate from the orbit into the interior part of the nerve. The significance of this finding is discussed particularly with regard to the pathogenesis of retrobulbar neuritis.