Leucocyte transendothelial migration (TEM) or diapedesis is pivotal in leucocyte trafficking during the inflammatory and immune responses. The endothelium plays an active role in this process, triggering an array of signalling pathways and reorganizing its cytoskeleton and membrane to facilitate leucocyte TEM. Diapedesis can occur between endothelial cells (paracellular) or through individual endothelial cells (transcellular). This latter route accounts for up to 30% of the total diapedesis in certain endothelial cell types in vitro. Mechanisms underlying both routes of diapedesis have been subjected to intense investigation during recent years. Here we describe a method to discriminate between the paracellular and the transcellular routes of diapedesis in vitro. The method is based on a transmigration assay of human T lymphoblasts through TNF-α-stimulated human primary endothelial monolayers, a triple fluorescence labelling of F-actin, the adhesion receptor ICAM-1 and the junctional protein β-catenin and a subsequent acquisition of z-stacks of high-resolution confocal sections.