Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat microvascular endothelium (MVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells (PAEC) and MVEC expressed PAR-1 and PAR-2, however only MVEC expressed PAR-3 by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH2 and TFLLRN-NH2) were employed to confirm a role for the PAR-1 receptor. PAR-APs (25 to 250 µg/mL) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in MVEC in contrast to PAEC. These results were confirmed in the isolated perfused rat lung preparation. PAR-APs (100 µg/mL) induced a 60% increase in the filtration coefficient, K_f over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro that corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.