Lymphocyte migration into the central nervous system is a central event in lesion formation in MS. Both interferon beta (IFNbeta) and copolymer-1 (Cop-1) reduce the overall lymphocyte entry into the brain through the blood-brain barrier (BBB) as judged by MRI based studies. In this study, we used a modified Boyden chamber assay in which human brain microvascular endothelial cell (HBEC) monolayers are grown on a fibronectin coated transwell membrane to evaluate in vitro migration of allo-antigen Th1 and Th2 lymphocytes across brain endothelium. We confirmed previous observations showing that migration rates of Th2 lymphocytes across HBECs were higher than migration rates of Th1 cells. When HBECs were pre-treated with IFNbeta (100 U/ml) 30 min prior to migration, the migration rate of Th1 was significantly decreased (45% reduction) while the migration of Th2 remained unchanged. Addition of Cop-1 (30 microg/ml) to HBEC monolayers 30 min prior to migration significantly increased the migration rate of Th2 cells and did not affect the migration of Th1 cells. We did not observe any changes in (1) the expression of adhesion molecules on the surface of HBECs and (2) the pattern of chemokine production by HBECs after IFNbeta or Cop-1 treatment. The changes in cellular migration rates were not paralleled with changes in diffusion of large molecular weight tracers across brain ECs. Our data support the notion that immuno-modulators used for the treatment of MS selectively and differentially regulate the migration of T helper lymphocyte subsets and that Cop-1 promotes trans-endothelial migration of Th2 cells across the BBB.