The facilitative glucose transporter GLUT1 is abundant in cells of the blood-ocular barrier and serves as a glucose transport mechanism in the barrier. To see the relationship between the glucose transfer function and junctional proteins in the barrier, we examined the localization of GLUT1 and the tight junction proteins, occludin and ZO-1, in the mouse eye. Their localization in the retina, ciliary body, and iris was visualized by double-immunofluorescence microscopy and immunogold electron microscopy. Occludin and ZO-1 were colocalized at tight junctions of the cells of the barrier: retinal pigment epithelial cells, non-pigmented epithelial cells of the ciliary body, and endothelial cells of GLUT1-positive blood vessels. Occludin was restricted to these cells of the barrier. ZO-1 was found, in addition, in sites not functioning as a barrier: the outer limiting membrane in the retina, in the cell border between pigmented and non-pigmented epithelial cells in the ciliary body, and GLUT1-negative blood vessels. These observations show that localization of occludin is restricted to tight junctions of cells of the barrier, whereas ZO-1 is more widely distributed.