Endothelial cells control water and solute exchange between the intravascular- and the interstitial space by a transcellular and paracellular pathway. In the regulation of both permeability pathways caveolin-1, the main scaffold protein of caveolae seems to be critically involved. We show that caveolin-1 plays a dual role regarding the paracellular and transcellular barrier function of endothelial cells. Using caveolin-1 negative endothelioma cells generated from caveolin-1 deficient mice or siRNA of caveolin-1 we demonstrate a critical role of caveolin-1 in the down regulation of the paracellular endothelial barrier function after thrombin stimulation by dynamin II-dependent endocytosis of junctional proteins. In contrast, in caveolin-1 negative endothelioma cells the constitutive endocytosis of albumin was two fold enhanced compared to caveolin-1 wt cells or after re-expression of caveolin-1 in caveolin-1 negative cells by lentiviral gene transfer. These data indicate that caveolin-1 has a dual role on the endothelial barrier function. It is required for the opening of intercellular junctions but it negatively regulates albumin uptake and in turn the transcellular albumin transport in endothelial cells.