In the current study, we assessed the effects of chemical inhibitors shown to be selective for protein kinase C (PKC) isoforms on lung barrier function both in vitro and in vivo. Rottlerin, a purported inhibitor of PKC, but not other chemical inhibitors, dose dependently promoted barrier dysfunction in lung endothelial cells in vitro. This barrier dysfunction correlated with structural changes in focal adhesions and stress fibers, which were consistent with functional changes in cell stiffness. To determine if the effects noted in vitro correlated with changes in intact lungs, we tested the effects of rottlerin in the formation of pulmonary edema in rats using both ex vivo and in vivo models. Isolated, perfused lungs demonstrated a significant increase in filtration coefficients (K_f) upon exposure to rottlerin, as compared to vehicle treated lungs; an effect that correlated with increased extravasation of Evan's Blue dye (EBD)-conjugated albumin. Additionally, compared to vehicle the ratio of the wet lung weights to dry lung weights was significantly greater upon exposure of animals to rottlerin; rottlerin also produced a dose dependent increase in EBD extravasation into the lungs. These effects on lung edema occurred without any increase in right ventricular pressures. Microscopic assessment of edema in the ex vivo lungs demonstrated perivascular cuffing, with no evidence of septal capillary leak, in rottlerin exposed lungs. Taken together, rottlerin increases barrier dysfunction in pulmonary EC monolayers and causes pulmonary edema in rats; results suggestive of an important role for PKC in maintaining lung endothelial barrier function.