A new model system for characterizing the human brain capillary, which makes up the blood–brain barrier (BBB) in vivo, is described in these studies and is applied initially to the investigation of the human BBB insulin receptor. Autopsy brains were obtained from the pathologist between 22–36 h postmortem and were used to isolate human brain microvessels which appeared intact on both light and phase microscopy. The microvessels were positive for human factor 8 and for a BBB-specific enzyme marker, γ-glutamyl transpeptidase. The microvessels avidly bound insulin with a high-affinity dissociation constant, KD= 1.2 > 0.5 nM. The human brain microvessels internalized insulin based on acid-wash assay, and 75% of insulin was internalized at 37°C. The microvessels transported insulin to the medium at 37°C with a t1/2=$70 min. Little of the 125I-insulin was metabolized by the microvessels under these conditions based on the elution profile of the medium extract over a Sephadex G-50 column. Plasma membranes were obtained from the human brain microvessels and these membranes were enriched in membrane markers such as γ-glutamyl transpeptidase or alkaline phosphatase. The plasma membranes bound 125I-insulin with an ED50= 10 ng/ml, which was identical to the 50% binding point in intact microvessels. The human BBB plasma membranes were solubilized in Triton X-100 and were adsorbed to a wheat germ agglutinin Sepharose affinity column, indicating the BBB insulin receptor is a glycoprotein. Affinity cross-linking of insulin to the plasma membranes revealed a 127K protein that specifically binds insulin. These studies demonstrate (a) the biochemistry of the human BBB may be investigated using the human brain microvessel model system, and (b) the human BBB insulin receptor has structural characteristics typical of the insulin receptor in peripheral tissues and may be part of a combined endocytosis-exocytosis system for the transport of the peptide through the BBB in man.