Leukocyte transmigration occurs at specific locations (portals) on the endothelium, but the nature of these portals is not clear. Using intravital confocal microscopy of anesthetized mouse cremaster muscle in combination with immunofluorescence labeling, we showed that in microvessels transmigration is mainly junctional and preferentially occurs at tricellular endothelial junctional regions. Our data suggest that enrichment of ICAM-1 near approximately 43% of these junctions makes these locations preferred for transmigration by signaling the location of a nearby portal, as well as preparing the endothelial cell (EC) junctions, to accommodate leukocyte passage. Blockade of the extracellular domain of the ICAM-1 significantly reduced transmigration (by 68.8 + or - 4.5%) by reducing the ability of leukocytes to get to these portals. In contrast, blockade of the cytoplasmic tail of ICAM-1 reduced transmigration (by 71.1 + or - 7.0%) by disabling VE-cadherin rearrangement. Importantly, venular convergences are optimally equipped to support leukocyte transmigration. Differences in EC morphology result in a significantly higher number of tricellular junctions in convergences compared with straight venular regions (20.7 + or - 1.2 versus 12.43 + or - 1.1/6000 microm(2), respectively). Consequently, leukocyte adhesion and transmigration are significantly higher in convergences compared with straight regions (1.6- and 2.6-fold, respectively). Taken together, these data identify an important role for EC morphology and expression patterns of ICAM-1 in leukocyte transmigration.