Pathways traversed by peripherally administered protein tracers for entry to the mammalian brain were investigated by light and electron microscopy. Native horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) conjugated to peroxidase were administered intranasally, intravenously, or intraventricularly to mice; native HRP was delivered intranasally or intravenously to rats and squirrel monkeys. Unlike WGA-HRP, native HRP administered intranasally passed freely through intercellular junctions of the olfactory epithelia to reach the olfactory bulbs of the CNS extracellularly within 45-90 minutes in all species. The olfactory epithelium labeled with intravenously delivered HRP, which readily escaped vasculature supplying this epithelium. Blood-borne peroxidase also exited fenestrated vessels of the dura mater and circumventricular organs. This HRP in the mouse, but not in the other species, passed from the dura mater through patent intercellular junctions within the arachnoid mater; in time, peroxidase reaction product in the mouse brain was associated with the pial surface, the Virchow-Robin spaces of vessels penetrating the pial surface, perivascular clefts, and with phagocytic pericytes located on the abluminal surface of superficial and deep cerebral microvasculature. Blood-borne HRP was endocytosed avidly at the luminal face of the cerebral endothelium in all species. WGA-HRP and native HRP delivered intraventricularly to the mouse were not endocytosed appreciably at the abluminal surface of the endothelium; hence, the endocytosis of protein and internalization of cell surface membrane within the cerebral endothelium are vectorial. The low to non-existent endocytic activity and internalization of membrane from the abluminal endothelial surface suggests that vesicular transport through the cerebral endothelium from blood to brain and from brain to blood does not occur. The extracellular pathways through which probe molecules enter the mammalian brain offer potential routes of passage for blood-borne and air-borne toxic, carcinogenic, infectious, and neurotoxic agents and addictive drugs, and for the delivery of chemotherapeutic agents to combat CNS infections and deficiency states. Methodological considerations are discussed for the interpretation of data derived from application of peroxidase to study the blood-brain barrier.