The conversion of prion protein (PrP^C) to its protease-resistant isoform is involved in the pathogenesis of prion diseases. Although PrP^C is highly expressed in neurons and other cell types, its physiological function still remains elusive. Here, we describe how we evaluated its expression, subcellular localization and putative function in brain endothelial cells, which constitute the blood-brain barrier. We detected its expression in microvascular endothelium in mouse brain sections and at intercellular junctions of freshly isolated brain microvessels and cultured brain endothelial cells of mouse, rat and human origin. PrP^C co-localized with the adhesion molecule platelet endothelial cell adhesion molecule-1 (PECAM-1); moreover, both PrP^C and PECAM-1 were present in raft membrane microdomains. Using mixed cultures of wild-type and PrP^C-deficient mouse brain endothelial cells, we observed that PrP^C accumulation at cell-cell contacts was probably dependent on homophilic interactions between adjacent cells. Moreover, we report that anti-PrP^C antibodies unexpectedly inhibited transmigration of U937 human monocytic cells as well as freshly isolated monocytes through human brain endothelial cells. Significant inhibition was observed with various anti-PrP^C antibodies or blocking anti-PECAM-1 antibodies as control. Our results strongly support the conclusion that PrP^C is expressed by brain endothelium as a junctional protein that is involved in the trans-endothelial migration of monocytes.