Osmotic opening of the blood-brain barrier had no effect on the structure of the interendothelial tight junctions located within approximately 9 micron 2 of brain capillary endothelial plasma membrane (junction-containing) examined in this study. These tight junctions restrict the passive diffusion between the blood and the brain and constitute the anatomic basis of the blood-brain barrier. Increased permeability of the blood-brain barrier in the cerebral cortex of the right hemisphere of rats, induced by an infusion of a hypertonic solution of arabinose and monitored with the protein tracer horseradish peroxidase (HRP), was evidenced by the extravasation of the tracer into the extracellular compartment of the brain. Freeze-fracture analysis of the capillaries from the same tissue revealed no alterations in the intramembrane components of the endothelial tight junctions. The junctions, which consist of 8-12 highly anastomosed parallel ridges situated on the PF fracture face of the endothelial plasmalemma, showed no loss of ridge continuity or intra-ridge connections, and were identical to zonulae occludentes from control capillaries. Consistent labeling of numerous vesiculo-tubular elements by HRP in the endothelia of experimental tissue and the three-dimensional nature of these elements observed in platinum replicas support the interpretation that these structures represent transendothelial conduits which are continuous with the luminal and abluminal surfaces of the endothelial cells. Absence of similar structures in control endothelia is taken as evidence that their presence in experimental tissues is a direct response to the osmotic insult. It was concluded, therefore, that during osmotic opening of the blood-brain barrier passage of HRP across the endothelium of brain capillaries is not by an inter-endothelial route due to disruption of tight junctions but rather by a transendothelial route due to amplified vesicular activity.