Mechanisms guiding migration of neutrophils through endothelium are poorly understood. We showed previously that CD31-CD31 binding acted as an ‘accelerator’ for neutrophils migrating on platelets, while neutrophil αvβ3-integrin acted as a sensor to align migration with the direction of imposed flow. Here, we perfused neutrophils over human umbilical vein endothelial cells (HUVEC) treated with tumour necrosis factor-α, and characterised the kinetics of migration over, through and underneath the HUVEC. Before penetrating the monolayer, activated neutrophils migrated relatively slowly over the surface (∼6 µm/min), preferentially in the direction of flow. Once transmigrated, neutrophils moved more rapidly (∼14 µm/min) without preferred direction. Treatment of HUVEC and/or neutrophils with function-blocking antibodies against CD31 reduced directionality but not velocity of migration on top of HUVEC, and reduced velocity of migration underneath the monolayer. If neutrophils were pre-activated with formyl peptide, they did not migrate through the HUVEC, but migrated with increased velocity and directionality on top. Under these circumstances, both velocity and directionality were reduced by blocking CD31. αvβ3-integrin did not regulate migration under any conditions. We conclude that CD31-CD31 bonds act as robust sensors which can guide neutrophil migration, and also modify its velocity. Thus mechanical and adhesive signals can regulate neutrophil migration driven by locally-acting chemotactic agents.