The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co-culture with astrocytes compared with endothelial cells cultured alone. In order to obtain reproducible and meaningful protein profiles of in vitro blood-brain barrier models, three sample preparation procedures were carried out to provide the first 2-D comparative proteomic study of BBCEC. Our study highlights advantages and drawbacks of each procedure. The cellular proteins prepared from mechanical scraping of collagen-seeded BBCEC were strongly contaminated by serum proteins. Enzymatic dissociation of BBCEC by trypsin or collagenase solved this problem. A comparative 2-DE profile study of collagenase-harvested BBCEC revealed that cytoskeleton-related proteins (actin, gelsolin and filamin-A) show the most significant quantitative changes in the Triton soluble protein fraction from BBCEC that exhibit characteristics closest to the in vivo situation.