To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the lumenal to the ablumenal compartment was time-dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and by ascorbate itself. Since the latter effect occurred over the physiologic range of ascorbate plasma concentrations, they could reflect a role for the vitamin in control of endothelial barrier function in vivo.