Penetration of immunoglobulins and/or migration of activated lymphocytes into peripheral nervous system (PNS) parenchyma are the initial key steps to develop immunological disorders of PNS including Guillain-Barre Β΄syndrome, IgM neuropathy and chronic inflammatory demyelinating polyradiculoneuropathy. Hence, it is important to know the cellular property of endothelial cells of endoneurial tissue origin (PnMEC) because these cells constitute the bulk of the bloodnerve barrier (BNB). For this purpose, we developed a method to isolate and culture pure populations of PnMECs from bovine cauda equina. PnMECs were identified by their cobblestone appearance, immunoreactivity against Factor VIII/von Willebrand factor (vWF) antigen, and positive uptake of DiI-Ac-LDL. The glucose transporter type 1 (GLUT1) expression of these cells was rapidly down-regulated in vitro. Other than GM3(NeuAc) and GM3(NeuGc) as major glycosphingolipids, PnMECs comprise GM1, GD1a, GD1b and GT1b, which are shared by PNS parenchyma, and sialyl lactosaminyl paragloboside (SLPG) as minor species. Because bovine PnMECs proliferate rapidly and a large mass of cells could be obtained, this method should contribute to the biochemical analysis of surface molecules in PnMECs that might play a key role in the formation of BNB as well as in pathological conditions involving the PNS.