Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real time fluorescence imaging technique was developed to simultaneously track influx of Alexa-680 labeled albumin and genetically tagged EGFP-neutrophils (PMN) within the wound bed. Vascular permeability increased ~3-fold more rapidly than the rate of PMN influx reaching a maximum at 12 hrs, on the order of ~0.15% per minute versus ~0.05% per minute for PMN influx, which peaked at 18 hrs. Systemic depletion of PMN with antibody blocked their extravasation to the wound, but did not alter the increase in vascular permeability. In contrast, pretreatment with anti-platelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelial specific agonists VEGF or thrombin at 24 hours post wounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 hour and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling.