In extravasation of T cells, little is known about the mechanisms of transendothelial migration subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26–expressing (CD26^hi) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3⁺ T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 μg/ml inhibited 80–90% of migration of CD3⁺ T cells through unstimulated and interferon γ–stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase–contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 μg/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26^hi. Second, solid-phase–immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26^hi T cells when added to T cells at a high dose of 10 μg/ml. Finally, both anti-4C8–induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26^hi cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26^hi cells adherent to HUVECs.