Recent studies from our laboratory demonstrated the involvement of endothelial cell reactive oxygen species (ROS) formation and activation of apoptotic signaling in vascular hyperpermeability after hemorrhagic shock (HS). The objective of this study was to determine if (-)-deprenyl, an antioxidant with antiapoptotic properties, would attenuate HS-induced vascular hyperpermeability. In rats, HS was induced by withdrawing blood to reduce the MAP to 40 mmHg for 60 min followed by resuscitation for 60 min. To study hyperpermeability, we injected the rats with fluorescein isothiocyanate--albumin (50 mg/kg), and the changes in integrated optical intensity of the mesenteric postcapillary venules were obtained intravascularly and extravascularly using intravital microscopy. Mitochondrial ROS formation and mitochondrial transmembrane potential (DeltaPsim) were studied using dihydrorhodamine 123 and JC-1, respectively. Mitochondrial release of cytochrome c was determined using enzyme-linked immunosorbent assay and caspase-3 activity by a fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cells using proapoptotic BAK as inducer of hyperpermeability. Hemorrhagic shock induced vascular hyperpermeability, mitochondrial ROS formation, DeltaPsim decrease, cytochrome c release, and caspase-3 activation (P G 0.05). (-)-Deprenyl (0.15 mg/kg) attenuated all these effects (P < 0.05). Similarly in rat lung microvascular endothelial cells, (-)-deprenyl attenuated BAK peptide-induced monolayer hyperpermeability (P < 0.05), ROS formation, DeltaPsim decrease, cytochrome c release (P<0.05), and caspase-3 activation (P < 0.05). The protective effects of (-)-deprenyl on vascular barrier functions may be due to its protective effects on DeltaPsim, thereby preventing mitochondrial release of cytochrome c and caspase-3--mediated disruption of endothelial adherens junctions.