Transendothelial migration of cancer cells from the vasculature into tissue stroma, is a final step in the metastatic cascade, prior to formation of secondary tumors. Due to its role in 2-dimensional migration of cells on extracellular matrix proteins, we hypothesized that focal adhesion kinase (FAK) promotes transendothelial migration of cancer cells. AU-565 cells are weakly invasive metastatic breast adenocarcinoma cells that migrate through bovine lung microvessel endothelial cell monolayers. Electric cell-substrate impedance sensing detects a significant decrease in monolayer resistance upon addition of AU-565 cells. Immunofluorescence microscopy and filter-based migration assays demonstrate that this drop in resistance correlates with transendothelial migration. Transfection of AU-565 cells with FAK siRNA results in significantly diminished transendothelial migration of AU-565 cells within 15 h. Expression of the dominant negative FAK inhibitor FAK-related non-kinase (FRNK) also results in delayed AU-565 transendothelial migration, whereas over-expression of wildtype FAK does not impact transendothelial migration substantially. These results demonstrate that FAK affects the rate of a key step in the metastatic cascade. (c) 2006 Elsevier Inc. All rights reserved.