The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-beta1 (TGF-beta1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-beta1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-beta1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-beta1 showed that beta-catenin, plakoglobin, alpha-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-beta1, cells began separating; however, beta- and alpha-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-beta1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-beta1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.