We tested the hypothesis that tumor necrosis factor- (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in -actin. PMEM were transfected with a wild-type-, mutant (tyrosine¹⁹⁸ to phenylalanine¹⁹⁸ [Y198F])-, mutant Y218F-, or a mutant Y306F- -actin construct tagged with Enhanced Yellow Fluorescent Protein (EYFP--actin). The cellular compartmentalization of wild-type and mutant EYFP--actin was displayed using EYFP fluorescence of the tagged -actin. -actin was quantified for the EYFP-tagged and native -actin using Western-blot assay. The effect of the EYFP--actin on a cell junction protein was assessed by association of EYFP- -actin with -catenin using confocal microscopy and co-immunoprecipitation. The permeability of PMEM was assessed by the clearance rate of Evans Blue labeled albumin. The cellular compartmentalization of wild-type and mutant EYFP--actin was similar to the native -actin. Incubation of PMEM with TNF (100 ng/ml) for 0.5 hr resulted in increases in permeability to albumin and a decrease in association of the EYFP--actin with -catenin. However, the expression of the EYFP-Y198F--actin and EYFP-Y218F--actin prevented the effect of TNF on -catenin and barrier function. The vehicle, wild-type EYFP--actin and mutantY306F--actin had no affect on the response to TNF. The data indicate that TNF induces an increase in endothelial permeability which is dependent on tyrosine¹⁹⁸ and tyrosine²¹⁸ in -actin.