Morphological investigations on the permeability of intercellular junctions between ependymal cells and between capillary endothelial cells in the subcommissural organ (SCO) of the guinea pig have been carried out using freeze-fracturing and tracer experiments with horseradish peroxidase (HRP). The ependymal junction reveals a moderately developed network of tight junctional strands surrounding the tall ependymal cell. The apical portion of this junctional network tends to form nearly complete strands, whereas the basal portion usually shows irregular, fragmented strands often arranged in hairpin-like structures. The passage of intraventricularly infused HRP is blocked, leaving unstained areas, at the level of membrane fusions. At the same time the lateral intercellular space below the junction is densely stained, probably due to invasion from the basal side through adjacent ordinary ependymal junctions. The SCO capillary endothelium shows a high distribution density of pinocytotic vesicles. Vesicular transport of intravascularly injected HRP is observed, but no HRP penetration occurs through the endothelial junction. The active participation of vesicles in tracer movement is shown in preparations fixed before administration of HRP. Extravasation of this tracer is indicated to some degree in the SCO capillary, but permeability here appears to be comparable to that of ordinary brain capillaries. Accordingly, the SCO ependymal tight junction seems to form an effective barrier not to blood plasma or similar materials but to apically secreted substances, preventing them from spreading back into SCO intercellular spaces.