This study investigates the fine structural localization of a blood-brain barrier using horseradish peroxidase (HRP) as a tracer. HRP was injected intravenously into mice, and its distribution in the cerebral cortex was examined using electron microscopy. Brains were fixed either by immersion or vascular perfusion 10–60 minutes after injection. HRP was found in the lumina of blood vessels and in some micropinocytotic vesicles within endothelial cells, but none was found beyond the vascular endothelium. Micropinocytotic vesicles were few and did not appear to transport HRP, while tight junctions between endothelial cells likely prevented its intercellular passage. These findings suggest a "barrier" at the endothelium of cerebral vessels, preventing HRP from passing beyond the vascular endothelium. The study also compared the permeability of cerebral vessels to those in heart and skeletal muscle. Cerebral vessels were found to be distinctly less permeable to HRP than those in heart and skeletal muscle, with this difference attributed to structural differences in the junctions between endothelial cells. The tight junctions between endothelial cells in cerebral vessels were found to be more structurally tight than those in heart vessels, which may explain the reduced permeability. Additionally, the low frequency of vesicles associated with transport of materials across endothelia in cerebral vessels, and the absence of peroxidase discharge on the contraluminal side of cerebral vessels, further support the existence of a blood-brain barrier. The study also found that HRP was confined to the lumina of blood vessels and did not penetrate the extracellular spaces of the brain. This suggests that the vascular endothelium acts as a barrier to the passage of HRP, and that the tight junctions between endothelial cells are responsible for preventing its intercellular passage. The findings have implications for understanding the blood-brain barrier, particularly in relation to the permeability of cerebral vessels to a wide range of solutes. The study also highlights the importance of tight junctions in maintaining the integrity of the blood-brain barrier.This study investigates the fine structural localization of a blood-brain barrier using horseradish peroxidase (HRP) as a tracer. HRP was injected intravenously into mice, and its distribution in the cerebral cortex was examined using electron microscopy. Brains were fixed either by immersion or vascular perfusion 10–60 minutes after injection. HRP was found in the lumina of blood vessels and in some micropinocytotic vesicles within endothelial cells, but none was found beyond the vascular endothelium. Micropinocytotic vesicles were few and did not appear to transport HRP, while tight junctions between endothelial cells likely prevented its intercellular passage. These findings suggest a "barrier" at the endothelium of cerebral vessels, preventing HRP from passing beyond the vascular endothelium. The study also compared the permeability of cerebral vessels to those in heart and skeletal muscle. Cerebral vessels were found to be distinctly less permeable to HRP than those in heart and skeletal muscle, with this difference attributed to structural differences in the junctions between endothelial cells. The tight junctions between endothelial cells in cerebral vessels were found to be more structurally tight than those in heart vessels, which may explain the reduced permeability. Additionally, the low frequency of vesicles associated with transport of materials across endothelia in cerebral vessels, and the absence of peroxidase discharge on the contraluminal side of cerebral vessels, further support the existence of a blood-brain barrier. The study also found that HRP was confined to the lumina of blood vessels and did not penetrate the extracellular spaces of the brain. This suggests that the vascular endothelium acts as a barrier to the passage of HRP, and that the tight junctions between endothelial cells are responsible for preventing its intercellular passage. The findings have implications for understanding the blood-brain barrier, particularly in relation to the permeability of cerebral vessels to a wide range of solutes. The study also highlights the importance of tight junctions in maintaining the integrity of the blood-brain barrier.