The blood vessels of the brain have unique characteristics which regulate the passage of substances from the blood to the interstitial space of the brain. The special collection of selective permeability and specific transport of molecules through the vasculature of the brain is known as the blood-brain barrier (BBB). Our laboratory discovered a monoclonal antibody which reacted with blood vessels of the brain which possess a BBB but not the blood vessels of peripheral organs or areas of the brain without a BBB. Due to its spatial and functional relationship with the BBB, this antigen was named endothelial barrier antigen (EBA). These studies aimed to define some of the biological and biochemical characteristics of the EBA molecules. EBA was localized to the luminal plasma membrane of CNS endothelial cells (EC) possessing a BBB by electron microscopy. EBA expression is developmentally regulated and closely follows the maturation of the BBB. Injection of monoclonal anti-EBA results in specific, sustained binding to BBB EC. EBA is a BBB-specific antigen which probably has a role in the interaction of blood-borne cells or molecules with cerebral EC. Monoclonal anti-EBA may be useful for delivering drugs specifically to the BBB. The 25, 28 and 31KD EBA proteins were characterized by classical biochemical analyses. These analyses included amino acid composition, peptide mapping, investigation of posttranslational modifications, two-dimensional gel electrophoresis, and finally amino acid sequencing. Amino acid sequence data for two peptides from the 31KD EBA were identical to mouse histone H1 (Yang et al, 1987). The identity of the isolated proteins was also confirmed by Western blot analysis using monoclonal anti-H1 histone (Chemicon). The biochemical data generated were consistent with results one would anticipate for H1 histones. However, anti-EBA recognizes an antigen in the endothelial cell membrane distinct from histone H1. This antigen was solubilized by treating isolated cerebral microvessel membranes with the detergent sodium deoxycholate. The experiments performed will simplify the future isolation of the EBA protein(s).