The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510-522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487-491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time-dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate-mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK.