Brain capillary endothelial cells compose the blood-brain barrier, which has a crucial role in maintaining the normal extracellular environment of the central nervous system. We have developed a method to isolate endothelial cells from mouse brain and maintain them in a relatively pure primary culture. Using a subtractive hybridization technique, a novel cDNA, termed MBEC1 (mouse brain endothelial cell 1), has been isolated from the cultured mouse brain capillary endothelial cells. MBEC1 is a 1435-bp cDNA predicted to encode a protein of 218 amino acids. The predicted protein is similar to those of the newly characterized Clostridium perfringens enterotoxin receptors and is the mouse homolog of a recently described human cDNA clone, which is hemizygously deleted in individuals with velo-cardio-facial syndrome and DiGeorge syndrome. MBEC1 was expressed in our cultured MBEC, in freshly isolated MBEC, in a variety of mouse organs, and in mouse embryos as early as embryonic Day 7. In situ hybridization and immunocytochemical analyses revealed the presence of the MBEC1 mRNA and its protein product in brain capillary endothelial cells, as well as in a subset of other endothelial and epithelial cells. Moreover, developmental regulation of expression of MBEC1 was present in respiratory epithelium. Our research thus provides a new molecule for further study of the function of normal and abnormal blood-brain barrier as well as of other specialized endothelia and epithelia.